encyclopedia of analytical chemistry[1]

8/3/2019 Encyclopedia of Analytical Chemistry[1]

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NEAR-INFRARED ABSORPTION/LUMINESCENCE MEASUREMENTS 1

Near-infrared

Absorption/LuminescenceMeasurementsA.R. Swamy, J.C. Mason, H. Lee, F. Meadows,M. Baars, L. Strekowski, and G. PatonayGeorgia State University, Atlanta, USA

1 Introduction 12 Near-infrared Chromophores and Light

Absorption Properties 3

3 Chemistry of Near-infrared Dyes 43.1 Indocarbocyanine Dyes 43.2 Squarylium Dyes 73.3 Phthalocyanines and

Naphthalocyanines 74 Analytical Applications of Near-infrared

Fluorescence 84.1 Applications of Non-covalent Label-

ing with Near-infrared Dyes 94.2 Bioanalytical Applications of Cova-

lent Labeling with Near-infraredDyes 9

4.3 Environmental Applications of Near-infrared Fluorescence 21

4.4 Other Applications of Near-infraredFluorescence 24

5 Conclusions 29Abbreviations and Acronyms 29Related Articles 30References 30

The near-infrared (NIR) region (6001100 nm) offers several advantages in comparison with the conventional ultraviolet/visible (UV/VIS) region for spectroscopic measurements and detection. The lack of commercial instru-mentation impeded the development of NIRtechniques for years. The advent of inexpensive photodiodes and diodelasers used widely by the telecommunication industry gavethe necessary impetus for development of analytical NIRtechniques. This article describes the fundamental prop-erties of NIR uorophores and absorbers and provides acomparison with conventional UV/VIS dyes. The variousanalytical applications developed in the past decade that

have utilized the advantages offered by this region aredescribed with a particular emphasis on bioanalytical uses.

1 INTRODUCTION

The current interest in development of new tech-niques for analysis is largely attributed to limitationsof the more conventional instrumental methods. Classi-cal biomolecule identication usually involves separationof a complex mixture of biological molecules followedby tests for identication of the separated fractions.No single test provides denitive identication of anunknown biomolecule, hence a complex series of testsare required. Such processes are time-consuming andcannot be carried on a reasonable timescale needed inclinical laboratories. These obstacles and the desire toattain adaptability to primitive eld test conditions have

prompted many researchers to explore modern alter-natives to classical instrumental procedures. Most of these modern analytical tools are characterized by rapiddata acquisition and data reproducibility which is dueto coupling with computer-aided instrument control, datarecording and interpretation. A numberof modern instru-mental techniques have been applied to the identicationof biomolecules. These well-established methods includehigh-performance liquid chromatography (HPLC), cap-illary electrophoresis (CE), circular dichroism (CD), gaschromatography (GC), and mass spectrometry (MS).Many of these techniques have also been adapted foreld applications. Most of these techniques convention-ally have been applied in the UV/VIS region. However,the NIR region offers several advantages over the con-ventional UV/VIS region.

Sir William Herschel, the English astronomer, discov-ered the NIR region (6001100nm) at the beginningof the 19th century 1 by using a prism and a ther-mometer; however, this region of the electromagneticspectrum did not receive much attention until the adventand recent availability of inexpensive light sources,such as diode lasers and detectors such as photodi-ode and avalanche photodiode (APD) detectors. The6001100-nm region corresponds to an energy range of

4826kcalmol 1. Atomic and molecular transitions inthis long-wavelength region are processes that requirerelatively low-energy photons, because the ground- andexcited-state species are close in energy. As a result,several classes of molecules, such as polymethine andphthalocyanine (Pc) dyes, and certain elements, such asruthenium and osmium, require a moderately low energyinput to produce spectroscopically measurable electronictransitions.

Owing to its sensitivity and selectivity, uorescenceis used as a major analytical tool in the identicationof target molecules of interest. Typically, this involves

using uorophores as reporter molecules or labels. Back-ground uorescence from components other than the

Encyclopedia of Analytical ChemistryR.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd


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2 ELECTRONIC ABSORPTION AND LUMINESCENCE

Visiblefluorophores

PAHs

Biomolecules NIR dyes

Porphyrins

200 400 600 800 1000nm

Figure 1 Visible to NIRregionandpossible interferences fromnative uorescence.

uorophore of interest decreases the sensitivity of detec-tion in solution. In biological systems, this backgrounduorescence is typically from the autouorescence of certain biological components. Similar problems areencountered with environmental samples. Typical back-ground uorescence occurs at all wavelengths in thevisible region and at various intensities depending onthe concentration of interfering molecules present in thesample (Figure 1). Elimination of background uores-cence is essential where the undesired components arenot separated and uorescence must be detected in thepresence of intervening molecules. In uorescence anal-ysis, excitation light can be scattered from interactionwith various types of molecules at the surfaces of con-tainers. This scattered light effect is present in all typesof uorescence detection. Scattered light contributes toa signicant portion of background noise, especially inbiological samples, and it can be due to either Rayleigh,Raman, or Tyndall effects. These interferences can bebest minimized by using uorophores with relatively highStokes shifts (typically > 4050nm).

Fluorescence is more sensitive and selective thanabsorbance as a spectroscopic tool, not only because it ismeasured against a zero background but also because

the magnitude of uorescence signal, F , at low dyeconcentrations is expressed by Equation (1):

F D 2.303 f I 0e bC 1

where I 0 is theexcitationpower, e is themolar absorptivityat the excitation wavelength, f is the quantum yield, bis pathlength, and C is the dye concentration. It can beseen that the limits of detection can be improved by astronger excitation source. Laser-induced uorescence(LIF) provides a superior approach to improve thesensitivity of uorescence techniques. However,one mustkeep in mind that the limit of detection increases only

as the inverse square root of excitation power and astrong excitation source can cause photo bleaching of

Table 1 Comparison of NIR and visible laser excitationsources

Parameter Laser diode Argon laserWavelength (nm) 785 488Lifespan (h) 100 000 C 3000Power output (W) 0.02 15Power consumption (W) 0.15 1800Replacement ($) < 150 > 5000

the uorophore. The limitations of conventional lasersas excitation sources include their high price, size,maintenancecosts, andtheir limitedwavelengthselection.LIF in the NIR region (6001100nm) offers severaladvantages, and the recent advances in semiconductor

laser technology have made the use of lasers morepractical. In addition, the extensive use of NIR-emittinglaserdiodes in the telecommunications industry has madethem more readily available. These types of lasers areinexpensive (typically < $150), small ( 1 cm), and havea long operating lifetime ( > 100000h). A comparisonof selected visible and NIR laser excitation sources isprovided in Table 1. The galliumaluminumarsenide(GaAlAs) laser diode hasdrawnmuchinterest because itsemission wavelength of 785 nm is compatible with severalclasses of polymethine cyanine and naphthalocyanine(NPc) dyes that exhibit NIR uorescence. 2

In addition to the availability of diode lasers used forexcitation, uorescence detection in the NIR region hasadvantages since noise resulting from scatter is related towavelength ofdetectionby thefactorof 1 / l 4 . Detection at820 versus 500 nm results in more than a sixfold reductionin scatter noise. The low background interference in theNIR spectral region allows NIR uorophores to be usedas ideal probes in both biological and environmentalapplications. The advantages offered by the NIR regionare summarized in Table 2. Detection in the NIR allowsthe commonly used photomultiplier tube (PMT) to bereplaced with the more efcient photodiode and APDtype. The APDs have excellent quantum efciency in the

NIR region. Table 3 illustrates the advantages offered byAPDs in comparison with PMTs. These benets includetheir low cost, compact structure, and their durability. Inaddition, they have low internal noise and very low powerconsumption. All these features allow NIR uorescence

Table 2 Comparison of NIR and visible regions

Noise source NIR region Visible region

Detector Low HighScatter

(Rayleigh/Raman)Reduced 6 greater at 520nm

than 820 nmBackground/auto-

uorescenceMostly absent Autouorescence of

biomolecules


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Table 3 Comparison of APDs and PMTs

Parameter APD PMT

Replacement cost ($) < 50 500Lifetime (h) > 100000 10000, sensitive to

overexposureQuantum efciency

(at 820nm) (%)80 0.3

Internalamplication

High Low

Size Very small (nm) Small (cm)Power consumption Very low Low

instrumentation to be highly versatile and amenable tominiaturization. These features aid in the development

of a portable compact and rugged instrument for eldapplications.The recent progress in solid-state diode laser tech-

nology, the availability of commercial NIR absorbingdyes, and the development of diverse synthetic routesto functionalized NIR absorbing dyes have allowed NIRspectroscopy to be used in many diverse applications.These specic uses rely on the response of the chro-mophore after its excitation by NIR radiation. A simpleabsorber can be used in spectrophotometric analysis, 3as molecular probes in immunoassays (IAs), or in appli-cations of security printing, such as laser-readable barcodes. 4 The absorbercan re-emit theinput energyvia u-orescence or phosphorescence. Also, the NIR-absorbingchromophore can convert the excited-state energy intoother forms that can be utilized for specic applications.After absorption of laser energy, the chromophore canproduce a local heating effect in the medium in whichit resides. 3,4 This technique is used in the optical datarecording technologies, such as WORM (write once readmany) and DRAW (direct read after writing), 4 fullcolor laser imaging, and in medicinal uses such as tissuewelding. 3 Conversion of theNIR radiation into electricalenergy by the chromophore is the fundamental basis forphotovoltaiccellsandelectrophotography, includinglaserprinting. 3,4 Furthermore, transfer of the chromophoresexcited-state energy to endogenous ground-state molecu-lar oxygen is the foundation for the selective destructionof neoplastic tissues in photodynamic therapies. 4,5 Inparticular, the NIR region provides a convenient spectralrange for photodynamic therapies and in vivo imagingowing to the multicentimeter penetration of biologi-cal tissues. 5 Taken together, these benets make NIRanalysis an operator-safe, nondestructive, sensitive, andversatile technique that is taking the place of conven-tional methods, such as radiolabeling. Moreover, dilutesamples can be analyzed safely and relatively fast com-

pared with other techniques. Several applications havebeen developed in the past decade utilizing the various

advantages offered by this novel methodology rangingfrom DNA sequencing, pH and hydrophobicity deter-mination, metal ion detection, antibody (Ab) labeling,HPLC, and high-performance CE.

2 NEAR-INFRARED CHROMOPHORESAND LIGHT ABSORPTION PROPERTIES

To take full advantage of the high-technology NIR instru-ments, new NIR chromophores with specic propertiesare required. The comprehension of the colorstructurerelationship of near-infrared dyes (NIRDs) can provideuseful information for their development since the light-

absorption property of an organic molecule is correlatedwith its structural features. A highly conjugated system ineither a linear or a cyclic arrangement in the molecule isresponsible for the absorption maxima in the NIR region.

In principle, the absorption property of a chromophoreis the characteristic of theenergy that is absorbed to causethe electronic transition. NIR-absorbing chromophoresrequire relatively low energy for the transition, and thiscorresponds to the longer wavelength of the electromag-netic spectrum in comparison with UV/VIS absorption.Since the transition energy is the energy required for asingle electron to be excited from the highest occupiedmolecular orbital (HOMO) to the lowest unoccupiedmolecular orbital (LUMO), the closeness between theHOMO and LUMO orbital is primarily responsiblefor the amount of energy required to induce this elec-tronic transition. Therefore, the shift of the absorptionmaxima ( l max) into the NIR region can be induced if the gap between the HOMO and the LUMO orbitalsis brought close enough to give the transition energyin the range 4826 kcalmol 1. In practice, effectivestructural modication of the chromophore can causethe desired bathochromic shift. Grifths summarizedin detail the general strategies to develop new NIR-absorbing dyes using this approach. 3 These strategiesinclude (i) extending the conjugation of a chromophore,(ii) increasing the interaction between electron donorand acceptor groups within a chromophore, (iii) alteringthe electronegativity of atoms within a chromophore,(iv) introducing specic branching or bridging withina chromophore, (v) metal complexation with a chro-mophore, (vi) intermolecular charge-transfer complexformation, and (vii) formation of a free radical that ispart of the chromophore.

The PariserParrPople molecular orbital (PPPMO)method 6 and XNDO/S method 7 are useful in the esti-mation of the absorption position of the chromophore

that needs to be constructed. The perturbational molec-ular orbital (PMO) theory in the form of Dewars rule


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0.11 0.123 0.11

0.043 0.0240.003

0 0

0.032 0.045*

Z 0.06 0.25 *0.06*

0.045 0.032

0.0430.0240.003

0

+

(1)

0

N

S

Et

S

NEt0.25

*

N

S R S

N+

** * * *I

(2)R = H, max = 758 nmR = CN, max = 860 nm ( = 102 nm)R = OMe, max = 730 nm ( = 28 nm)

Et Et

can also aid in the prediction of the light-absorptionproperties of chromophores with structural changes. 8This rule is particularly useful in the development of new NIRDs since one can correlate the spectroscopiceffects with a structural change at a specic position.In the basic analysis of an odd-alternate system of theconjugated chromophore, each alternate atom startingfrom a terminal donor is starred, as exemplied in (1) .Dewars rule implies that an increase in the electronega-tivity at a starred carbon results in a hypsochromic shift.On the other hand, a bathochromic shift will be inducedif the electronegativity of an atom at unstarred position isincreased. In particular, a bathochromic shift of a cyaninechromophore is induced if the electron donor groupsat starred positions or electron-withdrawing groups atunstarred positions are introduced. 6 This is further illus-trated by the studies of substituted trimethine cyanines(1) on the basis of the PPPMO theory by Yasui et al.,which showed the decrease in the electron densitiesat the starred positions and increase at the unstarredpositions of the polymethine chain accompanying therst excitation of (1) . 9 An additional example of theapplication of Dewars rule is given in (2) for three hep-tamethine cyanine dyes of a general structure (2) . As

can be seen, the electron-withdrawing substituent (CN)at the central unstarred position of the chromophorecauses a bathochromic shift and a hypsochromic shift isobserved for the electron-donating group (OMe) relativeto absorption of the parent unsubstituted dye R D H). 10

3 CHEMISTRY OF NEAR-INFRARED DYES

So far, the theoretical correlation between structure andabsorption properties has been discussed. These generalrules can also be applied in the practical synthetic design

of NIR-absorbing dyes. However, it should be notedthat not only the light absorption properties of NIR

chromophores but also other characteristics for differentanalytical applications are desired in the design of these systems. NIR chromophores with proper functionalgroups, organic and/or water solubility, high molarabsorptivity or uorescence maximum in the NIR regionare required for most applications, including medical andoptical purposes, dye lasers, and photographic sensitizers.These characteristics can be generally introduced intwo ways, namely (i) by the structural modication of commercially available dyes and (ii) by direct synthesisutilizing appropriate precursors. Several examples of direct syntheticroutes to NIRDs withspecicapplicationsare discussed below.

3.1 Indocarbocyanine Dyes

Functionalized NIR absorbing chromophores are widelyused for labeling purposes. However, the commer-cial availability of such systems is somewhat limited.The rst NIR-absorbing dyes containing ND CD S and

C(O)CH 2I functionalities for selective coupling withamino and thiol groups, respectively, of biomoleculeswere synthesized by Waggoners group. 11 These dyes,synthesized by using classical chemistry, contained thereactive group at one of the terminal heterocyclic units,and were extremely difcult to purify. Subsequently,Strekowski et al. 12,13 discovered a facile functionaliza-tion of indolium heptamethine cyanine dyes at the centralmeso position of the chromophore, and the synthesis of (8ac) is given in Scheme 1 as an example. These dyesare substituted with an isothiocyanato or N -succinimidylester function for selective labeling at the amino group of proteins or bioconjugates such as amino-functionalizednucleotides. 13,14 The structural design of (8) includes thepresence of sulfonic acid groups for excellent solubilityin water and the overall symmetry of the molecule tocontribute to the facile purication of the dye by simplecrystallization. The symmetry is responsible for a highmolar absorptivity of the chromophore. An importantstructural feature is the rigid trimethylene bridge in thecenter of the molecule that enhances the uorescencequantum yield by decreasing conformational freedomof the NIR uorophore in comparison with that of theuorophore without the bridge.

The key synthetic step to (8) is a facile nucleophilicdisplacement of the chloro substituent in the interme-diate product (7) which, apparently, involves an SNR 1pathway. 12 In the preparation of (7) the starting mate-rial (6) is derived from dialdehyde (11) (for structure,see Scheme 2) by the reaction with aniline. The secondcomponent (5) is prepared efciently by the reaction of (3) and (4) , as shown in Scheme 1.

This approach, as discussed above, was utilized byFlanagan et al. 15 in DNA sequencing involving capillary


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N

CH 3O

S+

i

N

CH 3

CH 3H 3C

+

Cl

N

O

O

CH 2CH 2CO

SO3

(4)(3)

(5)

iii

(7)(8a -c)

a X = S, R = NCS

b X = O, R = NCSc X = O, R =

O

O

CH 3

H 3C

NN

Cl

HH

Ph

(6)ii

Cl+

N

H3CH 3C

SO 3Na

N

CH 3CH 3

O 3S

X

N

H3CH3C

N

CH 3CH 3

O 3S

R

SO3Na

O

Ph

++

Scheme 1 Synthetic route to heptamethine dyes. (i) Reux in 1,2-dichlorobenzene. (ii) Reux in EtOH in the presence of sodiumacetate. (iii) Substituted sodium phenolate or thiophenol in DMF, 23 C. [Strekowski et al. 13 approach.]

N N

OX Y

O 3S SO3

(9a g)

X, Y X, Y

+

H, IH, BrH, ClH, F

Br, BrCl, ClH, H

abcd

ef g

gel electrophoresis (CGE). Their dyes (9ag) containdifferent atoms incorporated in the periphery of thechromophore, and have been prepared by substitutionof the meso -chlorine atom in the intermediate dyewith 2,5-disubstituted phenolates. The introduction of uorine or heavy atoms such as iodine, bromine, andchlorine into the molecular framework has been foundto affect the uorescence lifetime without affectingthe spectral properties of the chromophore. It wasreasoned that introduction of heavy atoms induces

spinorbit coupling. As a result, the intersystem crossing(ISC) rates are enhanced producing a reduction in

the excited-state lifetimes associated with the singletstate.

Recently, several commercial NIR-absorbing cyaninedyes have also been made available with amine-reactivegroups for covalent coupling with synthetically alterednucleotides or oligonucleotides. The dyes are avail-able under the Cy and IRD trade names, and havebeen synthesized by Mujumdar et al. 16 and LI-COR,Inc., 17 respectively. The LI-COR IRD dyes, such asthe nonsymmetric heptamethine dye IRD800 phospho-ramidite [ (18) , Scheme 2], can be prepared by using themethodology described by Narayanan et al. 18 The syn-

thetic route involves heating the bisaldehyde (11) andthe hydroxy-functionalized quatenary indolium salt (10)with azeotropic removal of water (Scheme 2) to givethe intermediate half cyanine carboxaldehyde (12) . Thisintermediate generated in situ is further allowed to reactwith indolium alkyl sulfonate (5) , which furnishes thecyanine dye (13) . The absorption maximum of the NIRchromophore in (13) can then be ne tuned by addi-tion of an electron-donating or electron-withdrawinggroup at the meso -position, as already discussed. Thus,the reaction of (13) with sodium phenolate (14) fur-nishes a phenoxy derivative (15) . Further treatment of

the meso -substituted cyanine dye (15) with 1H -tetrazole(16) and 2-cyanoethyl tetraisopropylphosphorodiamidite


6/35


7/35


processes. The dye-labeled dUTP analogs can beincorporated into DNA probes by nick translation,random priming, and polymerase chain reaction (PCR).

N

NH

O

O

O

NN+

O3S SO 3

(19)

Cy5-dUTP

O

OH

O P O P O P OO

O O

O O

O

NH

3.2 Squarylium Dyes

In additionto the indocarbocyaninedyes, synthetic routesto indolenine squaraine dyes, such as the N -succinimidylester derivative (23) (Scheme 3) for conjugation to pro-teins, are being developed. The squarylium dyes exhibit

high photostability and quite long uorescent lifetimes(nanosecond range) in aqueous solutions. Further bene-ts of using NIR-absorbing squaraine dyes for biologicalapplications include the fact that their spectral properties

(absorbance, emission, and lifetime) are independentof pH under physiological conditions (pH 69). 19 Asshown in Scheme 3, Terpetschnig et al. 19 synthesized awater-soluble NIR-absorbing dye containing an activatedester moiety. The reaction of (20) and (21) 20 pro-duced carboxy-substituted dye (22) . Following purica-tion by preparative thin-layer chromatography (TLC),the nonsymmetric squaraine (22) was coupled toN -hydroxysuccinimide to produce the active ester (23) .

3.3 Phthalocyanines and Naphthalocyanines

The NIR-absorbing dyes such as Pc and NPc type areused as industrial colorants and in special applications of electronics andoptics. 21 Theyefciently complexvarious

metal ions, and approximately 70 central metal ion speciesof Pcs are known. The cation is held in the planar 18-electroncavity andthe metal cationcan strongly inuencethe physical and spectral properties of the dye, includ-ing absorbance, emission, and uorescence lifetime. 21These NIRDs are sensitive to environmental changessuch as pH and metal concentration. Classical syntheticroutes to metal ion-containing Pcdyes [metal ionphthalo-cyanine (MPc) dyes] are illustrated in Scheme 4 wherethe metal ion acts as a central template for cyclotetramer-ization. As can be seen, MPc (28) is obtained directlyfrom diiminoisoindolenine (24) , phthalic anhydride (25) ,

phthalonitrile (26) , or phthalimide (27) in the presenceof a metal salt. 21 These dyes, though highly insoluble,are extremely stable and can be obtained by precipitationand further puried by sublimation. Metal ion-containing

OH

N

O 3S

N

Cl

O

ON

O

OO

(23)

N

SO3

HOOC

(20)

N

Cl

(21)

OH

N

HOOC

O 3S

N

Cl

O

(22)

i+

ii

OHO

O+

+

+

Scheme 3 Synthetic route to nonsymmetric squarine dye with N -succinimidyl moiety. (i) Reux in n-butanol and toluene withazeotropic removal of water. (ii) Stirring with N -hydroxysuccinimide and DCC in dimethoxyethane at 23 C. 19


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NH

NH

NH

O

O

O

CN

CN

NH

O

(24)

(25)

(26)

(27)

N

N

NN

N

N N

NM

(28)

i

ii

i

ii

O

Scheme 4 Classicalsynthetic routesto metal ionNPcs. (i) Heatin a high-boilingsolvent (e.g. quinoline) with metal salt. (ii) Heatin a high-boiling solvent with urea and metal salt.

NPcs, such as (31) in Scheme 5, are obtained in a similarfashion.The inherent sensitivity to metal and hydrogen ion

concentration makes Pcs and NPcs ideal candidatesfor use in pH or metal determination. However, thepoor aqueous solubility is a shortcoming that has to beovercome by structural modication or functionalization.

One approach to overcome the insolubility is to incorpo-rate sulfonate groups into the periphery of the existingPcs or NPcs (Scheme 5). Patonay et al. at GeorgiaState University 22 have synthesized a tetrasulfonylatedaluminum NPc (31) that can be incorporated into anoptical probe for NIR metal determinations. Once incor-porated into the ber, the four sulfonate groups at theperiphery of the NIR uorophore interact with metalions, such as K C , to produce spectral changes in theirabsorption maximum ( l max ) and/or uorescence inten-sity. The NIR probe studied by Patonay et al. providesmetal detection over the range 1 10 8 0.5 10 1 M.

4 ANALYTICAL APPLICATIONS OFNEAR-INFRARED FLUORESCENCE

LIF offers tremendous advantages over conventionalanalytical methods. The NIR region which has no back-ground interference when coupled with LIF can attainsensitivities obtained by radiolabeling. This methodalso provides a step towards the maximum achiev-able sensitivity, that is, single-molecule detection. Manyreviews of NIR uorescence that describe dyes, instru-mentation, and applications are available. An author-itative review, though dated, is provided by Warneret al. 23 A recent book summarizing the results of a

North Atlantic Treaty Organization (NATO) AdvancedResearch Workshop covers the synthesis, optical prop-erties and applications of selected NIRDs in high-technology elds. 24 In the next section, representativeexamples are reviewed to emphasize and acknowledgefurther the advantages and usefulness of NIR uores-cence techniques.

CN

CNN Al N

N

N

iii

(30)

(29)

N N

NNCl

N Al N

N

N

(31)

N N

NNCl

SO 3

SO3

SO3

O 3S

Scheme 5 Synthesis of tetrasulfonylated aluminum NPcs. (i) Reux in 1,2-dichlorobenzene containing 1 M AlCl 3. (ii) Heat inoleum (15% of free SO 3). 22


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4.1 Applications of Non-covalent Labeling withNear-infrared Dyes

The NIRDs used as reporter molecules can be attachedto the biomolecule of interest by two methods, via(i) covalent bond and (ii) noncovalent hydrophobicand/or ionic interactions. Theoretically, the hydropho-bic backbone of many NIRDs allows for noncova-lent labeling of large biomolecules. However, only alimited number of practical applications have beentested.

N

NaO 3S

N

SO 3

(32)

+

(33)

IN N

Cl

+

(34)

N N

Cl

+

NCS

SO 3

(35)

N N

R2

X

R1+

X = OPh-4(NCS), SPh-4(NCS); R 1 = Et, (CH 3)4SO 3Na;

R2 = Et, (CH 3)4SO 3

One of the earliest applications studied was the bindingof indocyanine green (ICG) (32) to serum albumins byKamisaka et al. in 1974. 25 Similar studies by Saudaet al. 26 using semiconductor laser uorimetry illustratedthat human serum albumin labeled with ICG providedpicomolar detection limits. A comparison of noncovalent

[(33) and (34) ] and covalent dyes (35) labeling usedin HPLC determination was investigated by Williams

et al. 27 in terms of stability and specicity. Their resultsindicated that noncovalent labeling occurs rapidly at aphysiological pH range. However, this interaction wasfound to be nonspecic and less stable than covalentbinding.

4.2 Bioanalytical Applications of Covalent Labelingwith Near-infrared Dyes

Many bioanalytical applications which rely on covalentlabeling have been developed. Techniques that utilizethis methodology include IAs, CE, DNA sequencing, andthe development of gene probes. 28 This section detailssome of these applications with their associated meritsand disadvantages.

4.2.1 Application of Near-infrared Fluorescence inCapillary Electrophoresis

Analytical separations of intact proteins using HPLCand CE have been shown to have excellent resolvingpower. 29 CE is particularly advantageous for proteinanalysis because of its higher chromatographic efciency,faster separations time, and ability to use small amountsof material. The low molecular diffusion rates of proteinshelp achieve theoretical plate numbers as high as 10 6.

Many excellent reference books on CE have appearedin recent years, 3032 and the reader is encouraged torefer to them for additional information not coveredbelow. The Handbook of CE 31 is very thorough, whilethe CE primer series offered by Beckman Instruments 30provides a brief overview of the principles of differentseparation techniques for a variety of applications.

Five main modes of electrophoresis have been devel-oped over the years. These include capillary zoneelectrophoresis (CZE), isoelectric focusing (IEF), CGE,isotachophoresis (ITP), and micellar electrokinetic chro-matography (MEKC). These different modes accountfor the wide applicability of CE to different analytes.However, one of the challenges in CE applications is thedetectionof analytesat lowconcentrations. This is a resultof the physical dimensions of the capillary used for sep-aration. Typical injection volumes in the nanoliter rangecombined with small detector windows of approximately100200 mm require highly sensitive detection systems.

UV/VIS absorbance is the most commonly useddetectionmethod forCE.The advantage is simplicity, lowcost, and ease of use. Detection limits are in the range of 10 13 10 16 mol for direct absorption (a factor of 10 lessfor indirect methods). Other methods employed includeMS, electrochemical, refractive index and radiometricdetection. 28 Fluorescence detection can greatly enhance

the sensitivity of CE methods. For example, directuorescence provides detection limits in the range of


10/35


10 15 10 17 mol (a factor of 10 less for indirect methods).However, the number of compounds that uorescenaturally is limited and derivatization of analytes witha uorescent tag is frequently required. Fluorescenceemission is measured against a zero background signal,resulting in improved detection limits in comparison withabsorption methods. Furthermore, LIF coupled to CEprovides a sensitivity of approximately 10 18 10 23 mol.This improved sensitivity of uorescence detection isattributed to concentrating high power monochromaticlight into a very small area, which provides an idealexcitation source for CE uorescence detection.

LIF is one of the most sensitive methods of detectionavailable for use with CE. Although the number of publications on the subject has been rising steadily each

year, the practical use of LIF with CE has been limitedowing to scatter from incident light reected by thecapillary walls with on-column LIF detection. Scattercan be greatly reduced by utilizing postcolumn detectionusing a sheath-ow arrangement, rst described by Chenand Dovichi. 33 However, this set-up adds a signicantamount of complexity to the method, requires additionalequipment such as a low-ow HPLC pump, and is notcommercially available. Another deterrent is the highcost associated with visible lasers which are suitablefor excitation of the more popular uorescent labelsincluding uorescein isothiocyanate (FITC) and 3-(4-

carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA).Several authors have reported on the advantagesof using semiconductor lasers with CE. Most inves-tigations to date have involved the use of far-reduorescent dyes. Williams et al. 34 described a diodelaser-based indirect absorbance detector for the anal-ysis of a series of tetraalkylammonium ions. In thisstudy, rhodamine 700 ( l abs D 642nm, l em D 668 nm) wasused as a background absorber, and detection limitswere found to be of the order of 20mM. Anotherstudy utilizing a time-resolved photon counting uorime-try CE method was described by Song et al. 35 Fouruorescent dyes were evaluated using an LIF detec-tor with a 74-ps pulsed semiconductor laser emittingat 655nm. The dyes investigated included methyleneblue ( l abs D 665nm, l em D 685 nm), oxazine 725 ( l abs D655nm, l em D 679 nm), 1,10,3,3,30,30-hexamethyl dicarbo-cyanine (HIDC) iodide ( l abs D 636nm, l em D 667nm),and rhodamine 700. The lowest limit of detection wasreported to be 2 fmol, which wasobtained for oxazine 725at a concentration of 0 .2 10 5 M. Fuchagami et al. syn-thesized a novel far-red uorescent labeling dye for usewith a semiconductor LIF detector. 36 Several aminoacids were derivatized with 9-cyano- N ,N ,N 0-triethyl-N 0-(50-succinimidyloxycarbonylpentyl)pyronine chloride

(l

abs D 663nm,l

em D 685 nm) and were separated anddetected by CE. The experimental system used a 2-mW

Table 4 Comparison of visible (R6G) and NIR (IR132)single-molecule detection. [Adapted from Soper et al. 38 ]

Parameter Visible NIRExcitation wavelength (nm) 532 800Observed wavelength (nm) 570 840Instrument response (fwhm) (ps) 100 250Photon detection efciency 0.0007 0.007Probe volume (pL) 1.1 0.8Transit time, t t (ms) 24 10Background rate in time window

(counts s 1)225 145 ( 12)

Average photons/molecule 39 18 ( 4)Detection efciency (%) 78 97 ( 7)Probability of error 0.3 0.04

semiconductor laser emitting at 660 nm and a PMTdetector. Detection was performed off-column using asheath ow cell to reduce Rayleigh scatter, achievinga limit of detection [signal-to-noise ratio (SNR) D 2] of 800 zmol.

The advantages of using APD CE detectors at wave-lengths above 650 nm have been reported. Kawazumiet al. 37 compared PMT and APD detection usingoxazine 725 excited by a 29-mW semiconductor laseremitting at 655nm. The APD was operated in a near-Geiger mode with time-gated detection, resulting in a47-amol limit of detection. The APD limit of detec-

tion was 98 times greater than that with a PMT. Asalready mentioned, detection in the NIR region offersconsiderableadvantagesover working in theconventionalUV/VIS region.

A signicant amount of work with NIR uorescent dyesincluding their applications to CE has been conducted atLouisiana State University by Soper et al. 3840 Theyobserved bursts of photons from single NIR uorescentmolecules in a owing stream using a Ti : sapphire laser(12 mW) and an APD detector. The dye infrared (IR)-132 (l abs D 805nm, l em D 847nm) at a concentrationof 25 fM in methanol was used in these experimentswith time-gated off-column detection. 38 A comparisonof a single-molecule detection of visible (R6G) dyeand the NIRD IR-132 (Table 4) shows a signicantreduction in background uorescence, resulting in alower discriminator threshold and error probability, andthereby provided a higher single-molecule detectionefciency in the latter case. A set-up similar to thephoton burst experiment, using continuous excitationand CE with on-column detection, was utilized for aninvestigation of binary solvent effects in CZE withseveral NIRDs and dye-labeled amino acids. 39 Oneof the dyes substituted with an isothiocyanate group(37) , was synthesized from dye (36) by Soper et al. and

covalently bound to amino acids. The results showedthat the detectability, efciency, and resolution of the


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N N

Cl

O 3SSO3

(36)

+

N N

O

O 3S SO3

(37)

+

NCS

Table 5 SNR, theoretical plates per meter and mass detectionlimits for cationic dyes in mixed methanolaqueous boratebuffers (pH 9.1). [Adapted from Flanaghan et al. 39 ]

Dye SNR Plates m 1 Massdetectionlimit (ymol)

95 :5 60 :40 95 :5 60 :40 95 : 5 60 :40

DTTCI 1478 65 500 5460IR-140 1713 146 64 500 63 600 446 20 280IR-132 1636 247 64 300 55 700 429 24 960HDITCP 1858 57 800 351

HDITCP, 1,1 0,3,3,30,30-hexamethyl-4,4 0,5,50-dibenzo-2,2 0-indotricarbo-cyanine perchlorate.

NIR methodology can be signicantly improved withthe use of mixed organic aqueous running buffers(Table 5). However, cationic NIRDs were found to beproblematic at low concentrations owing to capillary walladsorption. The on-column limit of detection was foundto be 0.4zmol using the dye HDITCP ( l abs D 780nm,l em D 825 nm) at a concentration of 2 10 10 M in 95%methanol, and the limit of detection of a dye-labeledamino acid was 21 zmol, using a 1 10 9 M solution in9 : 1 methanolwater buffer. A comparable CE systemwith a 10-mW Ti : sapphire laser emitting at 795 nmwas used to evaluate NIR uorescence detection forCGE and DNA sequencing applications. A comparisonbetween detection for NIRD-labeled primer (38) anduorescein labeled primer (39) is shown in Table 6.Under similar conditions, the signicantly lower Ramancontributions and background uorescence resulted in abackground of 10 000 counts for NIR compared with> 200000 counts for visible excitation in contrast to

the larger detection efciency at 800 nm in comparisonwith 550 nm. Although the NIRD exhibits a lower

quantum efciency, a much lower limit of detectionof 34zmol was achieved for an IRD41 labeled M13primer ( l abs D 786nm, l em D 812nm) in comparison with1.5amol for the visible primer primarily owing to thesignicantly smaller background in the NIR region. 40

Until recently, all near-infrared laser-induced uores-cence (NIRLIF) work completed to date involved the useof laboratory-made CE systems and detectors. However,two commercial LIF detectors are currently availablefor use with CE, manufactured by Beckman and ZetaTechnology. The Beckman LIF detector is specicallydesigned foruse with their P/ACE CE instrument, whilethe Zeta LIF detector is a general-purpose detector alsoapplicable to HPLCuse. Both detectors are optimized foruse in the visible region and utilize PMT detectors, with

laser excitation via a ber-optic cable. Recently, Patonayet al. at Georgia State University, actively involved inresearch in other areas of NIR uorescence, developed asimple interface between a commercial APD-based NIR-LIF detector and a CE instrument. The system retainedthe fully automated injection, separation, and data col-lection capabilities of the commercial CE instrument andwas optimized for detection around 820 nm with laserexcitation at 787 nm, where the detector sensitivity wasimproved about 400-fold in comparison with the conven-tional PMT-based LIF detector.

To illustrate the challenges in design of developinga NIR uorescence detector interface to exploit fullythe advantages offered by this region, a more detaileddescription of the work done by Baars et al. 28 ispresented in the following section. A Beckman LIFdetector with the P/ACE 5000 CE instrument wasmodied to accommodate the diode laser excitation andNIR uorescence detector. The back mounting plate anda portion of the self-aligning beam probe assembly of the Beckman LIF detector was used in the interface. Theat mirror, lter holder, PMT and detector electronicswere removed resulting in a collimated uorescenceoutput from the LIF cartridge, passing through the beamblock and beam probe, to allow interfacing with the NIRdetector. A proprietary microscope and laser assemblymanufactured by LI-COR, Inc. (Lincoln, NE, USA) wasused as the NIRLIF detector. This detector system iscomparable to components used in the commerciallyavailable automated DNA sequencing instrument 41 (LI-COR Model 4000), with the exception of modied focallength optics and an extra band-pass lter. The laserassembly for excitation contained a GaAlAs laser diodeemitting around 787 nm (20 mW peak power, modulatedwith a 50% duty cycle) and a focusing lens (focal length f D 46 mm). The detector consisted of a three-stagePeltier-cooled APD. The detector assembly contained a

plano-convex lens ( f D 31 mm) to collect the uorescenceimage, three identical band-pass lters (825 15nm)


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N NO

SO3NH

NH

O

P

S

OTGACCGGCAGCAAAATG

(38)

+

OO

O

COO

OHO

NHNO

PO

(39)

H

SO

OTGACCGGCAGCAAAATG

to reduce background noise from laser scattered light(Rayleigh), and a second plano-convex lens ( f D 31 mm)to refocus the signal on to the APD photoactive area(0.5 mm diameter). The interface between the LIFcartridge and the NIR uorescence detector comprisedan aspheric condenser lens mounted immediately pastthe beam probe assembly. An 800- m circular aperturewas installed at the focal point of the condenser lensto reduce stray light. The NIR detector was xed toan x yz micrometer stage installed on the mountingplate and focused on the image produced by the interfacelens. The diode laser was disconnected from the LI-COR

microscope assemblyandwas mountedon another x yzmicrometer stage mounted on the side of the CE base.The laser signal was focused directly on the excitationber, resulting in a 4 mW average excitation power atthe capillary interface. The optical path of the completesystem is shown in Figure 2. 28

Fluorescence emission was collected by the mirrorand reected as a collimated beam at 180 from theangle of excitation and the collimated beam was thenfocused by the aspheric condenser lens to the appropriateimage size, at the focal length of the detector. Thedetector optics lter the uorescence signal through

three band-pass lters prior to focusing the signal onthe window of the APD detector. Instrument control

Table 6 Electrophoretic parameters and detection limits forNIRD-labeled primer and uorescein-labeled primer.[Adapted from Williams and Soper 40 ]

Parameter NIR primer FITC primer

Migration time (s) 2522 660Apparent mobility (cm 2 V 1 s 1) 5.6 10 5 2.1 10 4Injection volume (L) 2 .9 10 9 1.1 10 8Amount injected (mol) 3 .8 10 19 5.8 10 17Net signal (counts s 1) 33 500 15 920Background (counts s 1) 10 000 19 490SNR 335 114Detection limit (mol) (SNR D 3) 3.4 10 20 1.5 10 18Quantum yield of tag 0.07 0.90

and data collection of the P/ACE 5000 CE systemwere fully automated through the Beckman SystemGold chromatographic software (version 8.01), run ona 486-33 personal computer. The detector signal wasinterfaced with the chromatographic software. This wasaccomplished by connecting theanalogdetector output toa Beckman406analog-to-digital convertor (ADC), whoseoutput can be collected by the chromatographic software.In their set-up, the P/ACE 5000 CE unit was used

to control all CE functions such as capillary rinses andinjectionandseparation parameters andin addition, it was


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LIF cartridge

CapillaryDiode

laser

Excitationfiber

0.8 mmslit Lens 1 Lens 2

APDdetector

Band-passfilters

LI-COR detectorInterface

Asphericcondenser

lens

Mirror

Inlet Outlet

Figure 2 Optical path of NIR/LIF interface with CE instrument.

used for data collection and chromatographic integration,reporting, and plotting functions. The maximum detectoroutput was matched to the 2-V input limit of the ADCusing a simple voltage divider circuit. A block diagram of the main instrument components is shown in Figure 3.

The image size produced by the aspheric condenserlens was evaluated for proper matching of the image sizeto the photodiode active surface area.The experimentallydetermined mean diameter is 0.575 mm, indicated aslight overlling of the 0.5-mm detector window. Therapid change in signal upon minor detector positionwas a good indication of an acceptable image sizematch. A three-dimensional representation of the imageproduced by the aspheric condenser lens is shown inFigure 4.

The sensitivity of the system was evaluated by injectingserial dilutions of the labeling dye NN382 (40) , (l abs D

406 A/Dconverter

Detectorelectronics

P/ACE5000CE

APDdetector

Diodelaser

Capillarycartridge

486-33 PCSystem gold

software

Figure 3 Experimental instrument component diagram.

776nm, l em D 796nm, H 2O) which is suitable for usewith the systems excitation and detection wavelengths(Figure 5). A linear detector response ( r D 0.9999) wasobserved over more than a 250-fold concentration rangebetween 1 .4 10 9 and5 .5 10 12 M (Figure 6).A SNR,

signal/root mean square noise) of 15 was observed fora 70-nL injection of the 5 .5 10 12 M solution, whichindicateda limit of detection(SNR D 3) of approximately80 zmol. The electropherogram of a 70-nL injection of the 5.5 pM solution (385 zmol injected) is provided inFigure 7.

The sensitivities obtained were under 100% aque-ous conditions, and additional improvements could beexpected with the use of organic modiers, cyclodex-trins, or micellar additives above their critical micelleconcentration in the run buffer, from an improved u-orescence quantum yield when the dye is exposed to amore hydrophobic environment. Comparison of the pho-tophysical properties of the dye in methanol suggestedthat a fourfold improvement in signal could be obtainedwhen using the dye NN382 in a more hydrophobic runbuffer, containing a cosolvent such as methanol. Addi-tional improvements in detection sensitivity could beattained using an NIRD with an emission wavelengthbetter matched to the band-pass lters in the detector.The current system utilizes three 825-nm band-pass l-ters that signicantly attenuate the uorescence signalemitted at 800 nm. The relative uorescence signal reach-ing the detector is only a fraction of the uorescenceproduced.

Light collection efciency and lens and lter transmis-sion efciency inuence the amount of light that will


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Image

X - a x i s ( m m ) Y - a x i s ( m m

)

R e s p o n s e

100.0

90.0

80.0

70.060.0

50.040.0

30.020.0

10.0

0.01.0

0.80.6

0.40.2

0.0 0.2

0.4 0.6

0.8 1.0

0.6 0.4

0.8

0.20.0

0.20.4

0.60.8

Figure 4 Three-dimensional representation of the image produced by the aspheric condenser lens.

N

O

(40)

SO3

NCS

+

SO3Na

N

NaO 3S

NaO 3S

actually reach the detector. Thus, the maximum ef-

ciency of the optical path in the experimental set-up canbe estimated as follows:

light collection efciency of mirror: 10%; transmission efciency of aspheric condenser lens:

98%; transmission efciency of rst detector lens: 98%; maximum transmission efciency of each band-pass

lter: 70%; transmission efciency of second detector lens: 98%.

The overall result is that only about 3.2% of theuorescence signal reaches the detector.

The quantum efciency of the detector (even thoughrelatively high for an APD at 800nm, 80%) further

700 720 740 760 780 800 820 840 860 880 900

0.010

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

100

200

300

400

500

600

700

800

900

1000

Wavelength (nm)

ExcitationEmission

A b s o r b a n c e

F l u o r e s c e n c e r e s p o n s e

0.00

0.10796 nm776 nm

Figure 5 Absorbance and uorescence spectra of NN382 (inphosphate buffer, pH 7.2).

reduces the number of photons that actually producea detector response. The improvement that can beattained when better matching band-pass lters with thedye emission prole, or dye selection with the systemband-pass lters, can be estimated. A comparison madebetween the area of the NN382 (40) emission curve from810 to 840 nm and the maximum area that could beattained from 781 to 811 nm ( 15 nm from the emissionmaximum)shows that maximumareawas70% largerthan

the area of the curve corresponding to the band-pass lterwavelengths. Using a dye with an emission wavelength


15/35


0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

10

20

30

40

50

60

7080

90

100201510

5

1000 200

Concentration (pM)

P e a

k a r e a

r e s p o n s e

Concentration (pM)

P e a

k a r e a r e s p o n s e

Figure 6 NN382 response curve (384 98 zmol on-column).

0 1 2 3 4 5 6 7 8 9 10 11

Time (min)

0.001

0.001

0.003

0.005

0.007

0.009

F l u o r e s c e n c e r e s p o n s e

( V )

NN-382

Figure 7 Electropherogram of 70-nL injectionof 5 .5 10 12 MNN382.

around 825nm could therefore result in about a 1.7-foldincrease in signal, assuming a comparable uorescenceemission prole. The noise would remain unchanged,suggesting that an additional 1.7-fold improvement inSNR could be attained. All the above-mentioned factorsdemonstrate important aspects of LIF instrumentationdesign. To optimize sensitivity, laser excitation shouldoccur below the absorption maximum when a dye has arelatively short Stokes shift. Band-pass lters should beoptimized for the uorescence emission maximum of thedye. This design will effectively attenuate the Rayleighscatter from the laser while optimizing uorescence signalcollection.

The automated NIRLIF system developedhas sensitiv-ity in line with state of the art results obtained by severalinvestigators utilizing highly optimized laboratory-madedetection systems. The latter systems utilize high-quality

microscope optics for uorescence emission collec-tion and tightly focused laser excitation beams. Few

investigators have reported limits of detection below thezeptomole range. Sub zeptomole detection limits havebeen achieved using CE by investigators including Soperet al. 38 (0.4 zmol) and Dovichi et al. 33 (0.050 zmol).Impressive sensitivities as low as single-molecule detec-tion have also been reported for molecules in a owingstream, demonstrating the possibilities of LIF when usedwith CE. 42,43 In order to attain these sub zeptomolelevels, an off-column sheath-ow detection arrangement

was generally utilized to reduce the amount of back-ground scatter. Neat dye solutions were used underconditions that optimized the quantum yield of the dye( ), not necessarily desirable for real CE applications.High laser powers, time gating and data smoothing havealso been utilized to achieve these results.

The data presented here are for an NIR labeling dyeused under 100% aqueous conditions in a fully auto-mated instrument, suitable for routine use. Additionalimprovements in sensitivity could be achieved by increas-ing laserexcitation power, improving the dyeuorescencequantum yield, and optimizing the dye emission prolewith the band-pass lters. The NIRLIF system uses morecompact, rugged, and less expensive components withan expected lifetime of > 100 000 h. The fully automatedinjection, separation, and data collection capabilities of the commercial CE system were preserved. 28

The fully automated system developed was then usedto evaluate the suitability of the NIRD NN382 (40) as apeptide labeling agent. Six angiotensin-I (Ang-I) variantswere selected as model peptides for derivatization andseparation studies. 44 The calculated charge-to-massratios of the derivatives and excess dye (at pH 7.2) aregiven in Table 7, listed in expected order of elution ina normal polarity CZE separation. The smallest charge-

to-mass ratio difference is between the human and Val-5forms, where the substitution of valine for isoleucine


16/35


Table 7 Sequence of labeled Ang-I peptides listed in order of decreasing charge to mass ratio and expected elution order in anormal polarity CZE system (amino acid differences from the

human form are bold and underlined)Ang-I variant Sequence Charge/mass

Goosesh (NN382)- Asn -Arg-Val-Tyr-Val -His-Pro-Phe-His-Leu

0.001237

Elasmobranch (NN382)- Asn -Arg- Pro -Tyr-Ile-His-Pro-Phe- Gln -Leu

0.001274

Salmon (NN382)- Asn -Arg-Val-Tyr-Val -His-Pro-Phe- Asn -Leu

0.001289

Human (NN382)-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

0.001664

Val-5 human (NN382)-Asp-Arg-Val-Tyr-Val -His-Pro-Phe-His-Leu

0.001674

Bullfrog (NN382)-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe- Asn -Leu

0.001732

NN382 0.003006

represents a difference of a single methyl group for the2295 (formula weight) labeled peptide.

The closely related decapeptides were labeled with theNIRD, separated using CE, and detected by NIRLIF.Derivatization of the peptides was achieved underaqueous conditions using 2.5 500pmol of Ang-I in a50-L sample (5 10 8 1 10 5 M). The uorescenceresponse was linear over a 200-fold range ( r > 0.9986)and the SNR of a 1.3-amol injection of the Ang-I variantswas > 15, indicating a limit of detection (SNR D 3) of < 240 zmol. Four of six peptides were resolved from eachother and excess dye using CZE. Two pairs of co-elutingpeptides were successfully resolved using MEKC. Twoneutral NIRDs were identied as suitable markers formeasuring electroosmotic and micellar ow. 44

AnotherapplicationevaluatedwiththeNIRLIFsystemwas in CGE. The suitability of NIRLIF detection withCGE was investigated. The purity of NIR-labeledprimerswas assessed, and detection linearity and sensitivityusing CGE were evaluated. Slab gel electrophoresis hasbeen used for many years for the analysis of proteins,oligonucleotides, DNA, and other biomolecules. Theapplications of this technique are very powerful andwidespread, but also slow, labor intensive and prone topoor reproducibility. 45 CGE was developed to automateand simplify traditional gel electrophoresis methods. Theadvantages of using CGE include the ability to automatethe process fully, provide on-line quantitative detection,and use smaller sample amounts. 3032,45 The capillariesare superior at dissipating heat, allowing the use of highervoltages, resulting in improved efciencies and shorterrun times.

LIF detection has been successfully applied toDNA sequencing methods. Swerdlow et al. 46 reported

sequencing rates of up to 1000 bases per hour usinguorescent-labeled primers. Another scheme uses uo-rescent intercalating dyes, such as those marketed byBeckman, for the analysis of ds DNA with LIF detec-tion. Although the total sample throughput is still limitedusing CE compared with slab gel methods, this limitationis likely to be overcome. Through the use of one-channel,four-dye detection schemes and the development of multicapillary instruments, this limitation can be over-come. Beckmanhas recently introduced an eight-capillaryCE instrument for DNA analysis and Ueno and Yeungreported on the use of a 100 capillary bundle. 47

CGE with LIF detection was utilized to assessthe purity of NIR-labeled oligonucleotides. The CGEmethod showed great sensitivity, with limits of detec-

tion around 90 zmol and a 1000-fold linear dynamicrange. The number of theoretical plates obtained forthe primers exceeded 300 000. The excellent sensitivityand high separation efciency observed for the NIR-labeled primers indicate their suitability for many otherCGE applications. 28 Sensitivity results are comparableto those reported by Williams and Soper 40 for a similarNIR-labeled primer. The excellent sensitivity and sepa-ration efciencies obtained for the NIR-labeled primerssuggest they may be suitable for many of the other CGEapplications described earlier.

The use of uorescent-labeled primers in binding

studies of synthetic oligonucleotides, such as antisensetherapeutics, has been demonstrated. Vilenchik et al. 48monitored the hybridization of a phosphorothioate targetwith a complementary labeled DNA probe. Protein toDNA binding studies were shownby Xian et al. 49 usingaCE mobility shift assay with CGE. An assay to determinethe activity of a transcription factor (SpP3A2), obtainedfrom the nuclei of a single sea urchin egg, was based onthe difference in mobility of a free or bound uorescentlabeled DNA probe.

4.2.2 Application of Near-infrared Fluorescencein Immunoassays

IA is a method of analysis that relies on specicinteractions between an Ab and an antigen (Ag) tomeasure a variety of substances, ranging from complexviruses and microorganisms to simple pesticide moleculesand industrial pollutants. To observe and measure thisreaction, a label is introduced via a second Ab. Con-ventionally, this label consists of a radioactive isotope(radioimmunoasay (RIA)), an enzyme (enzyme-linkedimmunosorbent assay(ELISA)) ora uorescentmolecule(uorescence immunoassay (FIA)). The use of Abs asanalytical regents was rst reported in 1959 when Berson

and Yalow successfully demonstrated the measurementof picogram levels of human insulin in samples of body


17/35


uids by RIA. 50 Since then, various IAs for detectinghundreds of molecules of endogenous and exogenous ori-gin have been described. This technique proved to bereliable, fast, and very sensitive; many other RIAs havebeen developed for clinical and medical tests since then.The use of immunochemical techniques in the environ-mental eld was rst proposed in 1971 by Ercegovich, 51who suggested the use of immunological screening meth-ods for the rapid detection of pesticide residues and forconrming results of conventional analysis. An RIA forthe insecticides aldrin and dieldrin was the rst reportedIA for an environmental contaminant. 52 Although a fewRIAsstillexistinthemedicaleld,theyareseldomusedinenvironmental and food analysis because of the need forspecial handling and disposal of the radioactive materials.

Radiolabels were gradually replaced with enzyme labelsowing to the hazards associated with radioactive mate-rials. ELISA, which was rst introduced by Engvall andPerlman in 1971, 53 hasbecome perhaps themost popularIA format used in laboratories today. The modern diag-nosis of many diseases, especially infectious diseases, isalmost completely dependent on these assays. In diseasesof global importance such as acquired immunodeciencysyndrome (AIDS), 54 cysticercosis, 55 malaria, 56 lar-iasis, and schistosomiasis, 57,58 IAs play a key role inscreening and diagnosis.

Utilizing the advantages of uorescence over absor-

bance, conventional uorophores can be theoreticallycapable of detecting fewer than 10 6 molecules L 1 59 inconjugation with the highly specic activity providedby IAs. However, in practice, the high backgroundfrom the biological samples autouorescence, or samplereagents and scattering and quenching effects of solvents,limit the detection to about 10 10 molecules L 1. LIFprovides an alternative to improve the sensitivity of uorescence in IAs. The use of LIF in IAs has beenreviewed. 6062 The limitations of conventional lasers asexcitation sources are their high price, size, maintenancecosts and their limited wavelength selection. LIF in theNIR region (600 1100 nm) offers several advantages.Recent advances in semiconductor laser technology havemade the use of lasersmore practical. Thewidespread useof NIR-emitting laser diodes in the telecommunicationsindustry has made them more readily available. This typeof laseris inexpensive (typically < $150)andsmall( 1cm)and has a much longer operating lifetime ( > 100 000 h). 63

The GaAlAs laser diode has drawn much interestbecause its emission wavelength of approximately 800 nmis compatible with several classes of polymethine cyaninedyes, which exhibit NIR uorescence. Detection in theNIR region allows one to replace commonly used pho-todiodes by APD. The APDs have excellent quantum

efciency in the NIR region. Some of the advantages of APDs are that they aremuch cheaper,more compact, and

longer lasting. In addition, they have low internal noiseand very low power consumption. All these featuresmake the NIR uorescence IA highly amenable to minia-turization and can aid in the development of a portable,compact and rugged instrument for eld application.

Heptamethine cyanine dyes are a class of NIR uo-rophores that have been used for DNA sequencing, pHand hydrophobicity determination, metal ion detection,and Ab labeling. 63 They are ideal for labeling Absinvolving conjugation chemistry, e.g. the reaction of the isothiocyanato group (NCS) on the NIRD withthe primary amine groups on Abs. These dyes havehigh molar absorptivities (ca. 10 5 Lmol 1 cm 1), highquantum yields (2040%), relatively short uorescencelifetimes (5001000 ps) and are small ( 1000 Da). The

small size of these dyes in comparison with the targetmolecule allows for a high number of labels per Abwithout compromising Ag/Ab interactions. This implieshigherselectivity andsignalcoupled with lowbackgroundinterference, in addition to the previously discussedadvantages offered by the NIR region. Additionally, theuse of a solid matrix generates a stronger signal by con-centrating theuorescentmolecules andtherebyreducingquenching effects of the solvent. 2

Boyer et al. were the rst to demonstrate the feasibilityusing diode laser detection in the NIR region. 64 Theycompared the efcacy of the NIR assay method with the

conventional ELISA method. Williams65

constructed anNIRuorescencedetectorfordetectinguorescencein anIA format. The set-up developed is illustrated in Figure 8.The instrumentation comprised an excitation sourcecoupled with a ber-optic cable, a silicon photodiodeas detector, a sample-holding apparatus that could beadapted to hold various forms of solid support matricescoupled with a motor drive, and a data acquisitioninterface. The instrument could detect concentrations of 100 pmol of NIRDs. Using this set-up, the near-infrared

Powersupply

Powersupply

Amplifier A/Dboard

PowersupplyMotordrive

Laser

DetectorOptical

fiber

Figure 8 Block diagram of NIR uorescence detector.


18/35


uorescence immunoassay (NIRFIA) on a nitrocellulosematrix was capable of detecting 5 10 10 M humanimmunoglobulin G (IgG). The overall assay could beperformed in less than2.5 h in the absence of the substratedevelopment step which is employed in the conventionalELISA method. This NIRFIA allowed the detection of approximately 470 00 labeled Ab molecules.

This method developed, however, had its disadvan-tages. A high degree of scatter generated by themembrane, problems with nonspecic binding of theconjugate, and its lack of compatibility with the mostcommon format of modern IAs, the microtiter plateassay format, limit its practical use. In continuation of the development of solid-phase NIRFIA, the above-mentioned issues and others were addressed in a

study by Swamy.2

An NIRFIA was developed basedon the heptamethine cyanine dye NN382 (40) . Thedye used in this study had a high molar absorptivity(e D 180000Lmol 1 cm 1) and quantum yield ( D 0.59,for dyeAb conjugate). The isothiocyanate functionalityreacts selectively with the amino group of the Abs to forma stable thiourea bond. The presence of the sulfonatedgroups makes thedyehighlywater soluble,making it idealfor labeling Abs. Another advantage of the negativelycharged sulfonate groups on the dye is that it minimizesnonspecic binding to the solidmatrix (polystyrene(PS)).

A solid-phase NIRFIA was systematically developedin the following steps. A LI-COR 4200 prototype uo-rescence microscope 41 was coupled with an orthogonalscanner and was evaluated for optimum depth of view.Next, the optimum scanner operating conditions weredetermined in terms of gain, offset, and scan speeds tooptimize settings for maximum dynamic range and SNRand ensure reproducible scans. Appropriate modica-tions to the mounting of the scanner microscope and

scanning platform were made to allow the examinationof microtiter plates (Figure 9). The linear response of thedetector was veried with increasing gain for a given volt-age setting. A number of microtiter plates (solid matrix)were then evaluated in order to provide the one withminimum background noise. Once the reader had beencongured and the solid support had been augmented,the procedure for covalent conjugation of NIRD NN 382(40) to goat antihuman immunoglobulin G (GAHG) wassystematically optimized in terms of pH, temperature,time of reaction, and reactant molar ratio. The condi-tions that provided the maximum results were a pH of 10, an initial dye : Ab ratio of 1 : 125, a temperature of 25 C and a coupling time of 1h. These conditions are allcritical parameters since any deviation results in under-or overlabeling of the Ab, which, in turn, results in alow specic activity and detection ability. The absoluteconcentration of reactants was found to be critical for therate of the coupling reactions; therefore, in the optimiza-tion procedure, stock solutions of dye (10 mg mL 1) andAb (13 mg mL 1) concentrations were used. Althoughthe conditions for coupling other species of Ab were notoptimized, other conjugates were successfully preparedwith high specic activity. The optimum conjugation pro-cedure was dened by the conditions that produced anNIRD-labeled Ab with the highest specic activity in animmunosorbent assay for normal human immunoglobu-lin G (NHIgG).Thebest conjugatewasused to determinedetection limits for the assay and was able to detect2 10 11 M of NHIgG. This provided a sensitivity about10 times greater than that achieved by conventional labelsin similar assays. Finally, the assay was validated by 100%agreement with clinically diagnosed samples for schisto-somiasis and its cross-reactors. 2

Thermoelectric cooler

Avalanchephotodiode

Laser diode

ELISA plate

Focusing lens

Detectorfilter stack

Collection lens

IRanalyzer4000X

Motor drive

Figure 9 Model assembly to scan microtiter plates.


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One major disadvantage of this method is the lack of commercial instrumentation for the assay. Quanticationof the signal obtained in an image format is problematic.However, commercial software for quantifying on linesignals from DNA sequences in the NIR region isavailable (LI-COR). Adaptation of similar software formicrotiter plate scans would be immensely helpful. Themethod developed does have many advantages overconventional methods and the versatile nature of thescan bed does not limit this assay to the microtiter format.Technically, the scanner could scan any matrix, limitedonly by the physical dimensions of the scanner (ca. 23 cmand 61 cm long). This could allow for scanning multipleassays and 5001000-well plates. The assay is very quick,requiring less than 20 min for the assay itself, and requires

fewer steps than ELISA. The ability to detect lowerconcentrations of Abs would help in earlier diagnosisand an opportunity to test drugs against opportunisticinfectious agents at an earlier stage. The labeling of Abswith NIRDs provides an important advancement to IAsand the comparatively small size of the NIRD allows fora higher molar ratio of NIRD per Ab. This in turn yieldsa higher signal and allows for lower detection limits.The assay has also been validated for clinical samplesand could be further evaluated in the development of adiagnostic test.

In principle, various bioanalytical methods can bedeveloped using the NIR immunochemistry describedin the preceding section. To demonstrate the versatilityof the method, Swamy et al. developed two bioanalyticalapplications utilizing NIRFIA in collaboration with theState University of New York (SUNY) at Brooklynand the Department of Entomology and EnvironmentToxicology at the University of California, Davis, CA,USA. The rst application involved an NIR-labeled Abin the detection of extracellular Ag expressed on cells.This demonstrated the feasibility of using NIR-labeledAbs in clinical imaging applications such as NIR opticaltomography. The second application uses the NIRFIAas an analytical tool in environmental applications inthe quantitative analysis of two pesticides, bromacil andfenvalerate.

In the evaluation of the NIRD-labeled Ab for imagingapplications, the NIRD NN82 was coupled to monoclonalAb E48 (MAb E48) and the conjugate was evaluated ina direct assay to detect a 22-kD Ag expressed on thesurface of human squamous cell carcinoma line (HuSCC)A431. The specicity of binding was conrmed in acompetitive assay. The preliminary results showed goodsensitivity and specicity of the NIRD-labeled Ab. In theenvironmental application, the assay was evaluated in arapid tracer format competitive assay for the detection of

the pesticides bromacil and pyrethroid. In this assay, thespecic Ab captured on the surface of microtiter plate

well and the tracer with uorescent label compete withthe analyte for the Ab. The results obtained showed thatthe NIRFIA was at least as sensitive as ELISA, with bothassays detecting pesticides in the micrograms per liter(parts per billion) range. 2

The chemistry and the instrumentation of the NIR-FIA technique for environmental applications have beendeveloped, but are amenable to further miniaturiza-tion. Further optimizations would allow for the futureconstruction of compact instrumentation for analysiswhich could conduct fast and reliable assays at remotelocations. In continuation of this approach, prelimi-nary results obtained in our laboratory demonstratedthe feasibility of using the NIRFIA approach for anNIR/ber optic immunosensor (FFOI). This approach is

particularly useful for measuring small amounts of ana-lyte and can be automated and carried out in remotelocations. Several uorescent immunosensors have beenreported; however, lack of commercial instrumentationand labels limit the use of these techniques and mostapplications utilize UV/VIS dyes. The applications of the visible uorescent immunosensors described are allsusceptible to interference from biomolecules such asbilirubin and porphyrins. A comprehensive review of immunosensors has been published by Robinson et al. 66Danesvar 67 developed a NIR/FFOI fordetectionof traceamounts of human IgG and Legionella pneumophilia .They optimized the assay on different solid phases suchas poly(methyl methacrylate) (PMMA) and PS. ThePS coating method eliminated the activation processrequired with PMMA, thereby reducing the prepara-tion time by 18h; in addition, it also eliminated theprotein G step of the previous assay. The assay was car-ried out in a sandwich format (Figure 10). The set-upused to measure the signal (Figure 11) allowed concen-trations of 1011 M for IgG (SD D 0.13) and 0.5 ngmL 1for Legionella pneumophilia sera type 1 (LPS1) 67 to bedetected.These results werecomparable to those attainedby conventional ELISA. Another application using a sim-ilar format was developed by Evans 68 and provided acompetitive assay for the pesticide bromacil. A detectionlimit of 5 ppb was obtained, which was slightly higherthan that reported for ELISA (0.1 ppb). However, themethod had advantages since it did not require exten-sive sample preparation, the assay time was substantiallyreduced, and the assay could be adapted for remote siteanalysis. 68

The NIR/FFOI technique in principle is a exiblemethodology, which can be theoretically adapted todevelop assays for any compound for which Abs areavailable, including infectious agents, serum analytes,and environmental pollutants. This technique provides

a real-time analysis with automated readout capabili-ties, eliminating the need for operator intervention or


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Figure 10 Sandwich assay on ber-optic probe.

Laser diode 780 nm

Excitation Emission

Photodiode

Fiber sensitiveterminal

Digitalvoltmeter

Lock-in amplifier

Figure 11 Fiber-optic immunosensor set-up.

complicated sample preparation. Although the detectionoptics for the assay were originally constructed on a2 3 ft optical table, the set-up can be reduced in size.The preliminary sensitivity, selectivity, and simplicity of the assay are encouraging and further design renementand other applications should aid the development of versatile NIR/FFOI.

4.2.3 Application of Near-infrared Fluorescence inHigh-performance Liquid Chromatography

Another bioanalytical application developed using theadvantages of NIR uorescence is HPLC detectors. Inrecent years, the ability to measure ultralow levels of pharmaceuticals in biological matrices has presentedsignicant challenges. Using uorescence as a tool fordetection, sensitivities of picograms per milliliter havebeen achieved. Rahavendran and Karnes have describedthe advantages of LIF detection in HPLC. 69 Therehave not been many publications dealing directly withHPLC detection in the NIR region. Until recently, twogroups worked on the development of applications of NIR uorescence in HPLC, Winefordners group at the

University of Florida and Ishibashis group in Fukuoka,Japan.

One of the rst NIR uorescence-based HPLC detec-

tors was developed by Ishibashis group. They detected0.3pg of a carbocyanine dye, 70 with instrumentationcomprising a 3-mW diode laser emitting at 780 nm witha PMT cooled to 20 C for detection. They reported adetection limit of 1.9 pg at room temperature. Sternberget al. 71 of Beckman Instruments used a 2.5-mW diodelasercoupled with a commercial diode-array detector andachieved a detection limit of approximately 10 10 M fora carboxyl cyanine-labeled oligonucleotide. Winefordneret al. explored the detection of cyanine dyes in differ-ent instrumental congurations, 72 using a diode laserfor excitation with a PMT for detection. They achieved

detection of 46 000 molecules in a liquid jet uorescencespectrometer. Karnes et al. 73 used diode laser-inducedNIR uorescence for detection of ICG in plasma. Theydemonstrated a detectability of more than two orders of magnitude over absorbance.

Another NIR HPLC application was reported byKuklenyik, 74 who developed and compared twodetectors for NIR uorescence detection based on a

Lenses

Laser diode780 nmInterference filter

830 nmInterference filteror filter bank

Flow-through cell

Photodiode

Figure 12 Optical path of NIR/HPLC uorescence detector.


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HPLC

Flow-throughcell

Laser source20 mW

Amplifier

Detector

Serial link

Microcomputer

Digital voltmeter

Pump

Waste

12345

Figure 13 Experimental set-up of NIR/HPLC uorescencedetector.

N

O

(41)

SO 3Na

NCS

N +

NaO 3S

N N ClO 4

(42)

+

silicone photodiode and an APD. The merits of this typeofdetectorwerealso compared withthose of conventionalPMT detectors. An HPLC system (SSI gradient system)with a high-pressure pump capable of providing 500 psipressure controlled by an IBM computer was coupledwith an in-house developed excitation and detectionsystem. The experimental set-up of the excitation anddetection and the nal set-up are shown in Figures 12 and13. It comprised a silicon photodiode detector coupledwith a 24-L HPLC ow cell shown in Figure 12 and,in the set-up with an APD, a modied Model 4200LI-COR microscope 41 was used instead of the siliconphotodiode. Detector performance was evaluated withtwo NIRDs, IRD40 (41) and 1,1 0,3,3,30,30-hexamethyl-

4,40

,5,50

-dibenzoindotricarbocyanine perchlorate (42) ,rst without an HPLC column followed by subsequent

0.010.020.030.040.050.060.07

0.080.090.10

0 0.5 1 1.5 2 2.5

Eluent (MeOH) volume (mL)

S i g n a

l ( V )

Figure 14 Detection of threeconsecutive injectionsof IRD40 on a C 18 column.

runs with C 18 HPLC columns. Detector signals forthree consecutive injections of IRD40 at 10 10 Mconcentration are shown in Figure 14. Detection limitsof 1.22 10 11 and 5 .89 10 12 M were achieved for thetwo dyes and the numbers of dye molecules detectedwere calculated to be 36 500 and 17 600, respectively. 74The overall results showed that the less expensive, moreefcient photodiode was sensitive for detection in theNIR region. The detector was designed to collect signalsat 820 nm, and a better match of the dye emission withthe detector and better light collection efciency shouldhelp improve the sensitivity much more. Even in theearly stage of development in this direction, the NIR

uorescence method shows sensitivity comparable toor better than those of conventional methods. Furtherevaluations and modications could help achieve muchgreater sensitivities.

4.3 Environmental Applications of Near-infraredFluorescence

The fundamental principle involving the analytical appli-cations of dyes to study the microenvironment essentiallyinvolves the changes induced by the analyte that can bedirectly measured by spectral changes. These changesdepend on the concentration of the analyte and theinteraction between the analyte of interest and the dye.Typically these probes involve a cation-selective receptoreither as an integral part of the chromophores p -systemor covalently attached to the uorophore via an alkyltether. 7577 The coordination of the cation affects thespectral position of absorption and emission bands, molarabsorptivity, and, to a smaller extent, the uorescencequantum yield. The interaction of a metal ion with anorganic reagent might result in the enhancement of theuorescence or the quenching of the uorescence of theorganic reagent in the presence of an analyte. A statisti-cal study showed that about 89% of luminescent methods

are based on the enhancement of uorescence and about11% are based on quenching reactions.


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The investigation of toxic metal ions in theenvironmenthas been of growing interest in the past few decadesand the decreasing permissible limits for most of thesemetal ions set by major health organizations such asthe World Health Organization (WHO) and the UnitedStates Environmental Protection Agency (USEPA) haveprovided a tremendous thrust for the development of sensitive analytical techniques. Most of the publicationsdevoted to the detection of metal ions by means of spectroscopic techniques used UV/VIS probe moleculessuch as rhodamine, uorescein, 8-hydroxyquinoline, andtheir derivatives. 7883 Table 8 provides a list of some of the different uorescent sensors that have been used forthe determination of the most commonly studied metalions, namely Al(III), Be(II), Co(II), Fe(III), and Li C .

Unfortunately, spectral interference is signicant in thisregion. The use of NIRDs is a better alternative. 8490The low background interference observed for othermolecules in the NIR spectral region together with thelonger Raman shift offers better SNRs. Casay et al. 91investigated tetrasubstituted NPc NIRDs as potentialprobes for the determination of toxic metal ions. Theydeveloped the rst NIR optical probe that could detectlead, lithium, and cadmium at parts per billion levels 91and potassium at 0.566ppm. 92 More recently, Tarazi 93reported the detection of metal ions using three novel

NIRDs, namely TG 170 (43) , NN525 (44) , and JCM-15C5 (45) . The spectral characteristics of the three dyesare listed in Tables 911. They evaluated TG 170 for thedetection of Al(III) and Be(II), NN525 for the detectionof Fe(III) and Co(II) ions, and JCM-15C5 for thedetection of Li C in the presence of other interfering ions.

N N

Cl

(43)

CO 2HHO HO 2C OH

CF3CO 2

+

N N

(44)

O

O

2+

HO 2C SO3

+

Table 8 Different uorescent methods currently employed for detection of various metal ionsMetal ion Reagent Method a Detection limit (ppm)

Al(III) Alizarin Red S E 8 10 2Lumogallion E 3 10 2 3.2 10 18-Hydroxyquinoline E 5 10 3Morin E 2 .5 10 4 5 10 3N -Salicylidene-2-hydroxy-5-sulfoaniline E 8 10 5 8 10 2Superchrome Carnet Y E 8 10 4 1.6 10 12-Hydroxy-3-naphthoic acid E 1 .8 10 4 1.8 10 3

Be(II) 2-Ethyl-5-hydroxy-7-methoxyisoavone E 4 10 4 1.2 10 22-Hydroxy-3-naphthoic acid E 1 .8 10 4 1.8 10 3Morin E 4 10 4 1.6 10 3

Co(II) Al(III)Pontachrome BBC E 10 3

Al(III) Superchrome Blue Black Q 10 3 2 10 22-[2-Hydroxy-1-naphthyl]dithiocarbazic

acid-4-chlorobenzyl esterQ 10 1 1.1

1-(2-Pyridylazo)-2-naphthol E 5 .9 10 2Al(III)Pontachrome BBR Q 2 10 2 2 10 1

Fe(III) 4 0-(4-Methoxyphenyl)-2,2 0,200-terpyridyl Q 10 2 10 1Rhodamine B Q 12,20,200-Terpyridyl Q 10 2 5 10 1

LiC Dibenzothiazolylmethane E 5 10 25,7-Dibromo-8-hydroxyquinoline E 2 .5 10 1 2.51,4-Dihydroanthraquinone E 5 10 2 5 10 11,5-Dihydroanthraquinone E 5 10 2 5 10 11,8-Dihydroanthraquinone E 5 10 2 7 10 18-Hydroxyquinoline E 4 10 2 1

a E D enhancement of uorescence; Q D quenching of uorescence.


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N N

NH

O

OO

O

O

(45)I

+

Table 9 Spectroscopic characteristics of TG170 in varioussolvents

Solvent l max l em f Log e(nm) (nm) (%) (L mol 1 cm 1)

Dimethyl sulfoxide 819 822 0.167 4.96Methanol 805 812 1.30 5.40Buffer (pH 5.9) 791 803 2.48 5.99

Table 10 Spectroscopic characteristics of NN525 in varioussolvents


Acetonitrile 664 673 0.86 5.64Dimethylacetamide 672 681 0.92 5.74Dimethylformamide 672 681 0.81 4.67Dimethyl sulfoxide 676 688 0.73 4.69Methanol 663 670 0.87 5.72Tetrahydrofuran 669 676 0.91 5.76Water 661 666 0.03 5.38

Table 11 Spectroscopic characteristics of KVA22 in varioussolvents


Acetone 780 799 3.02 4.12Acetonitrile 776 799 1.58 4.83Dimethyl sulfoxide 793 809 2.03 4.66Methanol 777 780 1.52 4.81Methylene chloride 787 803 2.74 4.96

The hydroxy carboxy functionality of TG 170 (43) hasa known selective complexation with Al(II) and Be(II)ions. Using this dye, they reported a detection limitof 52 ppb for Al(III) and 24.6 ppb for Be(II) ion. Forthe NIRD NN525 (44) , only Fe(II), Fe(III), and Co(II)ions resulted in quenching of uorescence. Detection

limits of 3.54 ppb for Fe(III) and 1.55ppb for Co(II)were obtained. The calibration curves obtained for the

06 9 12 15

500

1000

1500

2000

2500

3000

3500

Concentration of Fe(III) (10 8 M)

F l u o r e s c e n c e

i n t e n s i t y

( a . u . )

Figure 15 Calibration curve for detection of Fe(III) withNN525.

1.5 1.67 1.88 2.15 2.51 3 3.75 5 7.5400

500

600

700

800

900

1000

Concentration of Co(II) (10 8 M) F l u o r e s c e n c e

i n t e n s

i t y

( a . u . )

Figure 16 Calibration curve for detection of Co(II) withNN525.

two metal ions are illustrated in Figures 15 and 16. Thecrown ether functionalized cyanine dye JCM-15C5 (45)was used for detection of Li C ions in the presence of other interfering ions. They obtained a linear plot of uorescence intensity for Li C amounts ranging from0.0347 to 0.222 ppb (Figure 17). A detection limit of 0.0743 ppb was reported. The stability constant for theJCM-15C5 complex was higher than those achievedin other studies. The method development representsa signicant improvement over conventional methods.Owing to the minimal interferences encountered inthe NIR region, it eliminates the need for extensivesample preparation, and additionally it allows for thedevelopment of tests that can be carried out at remotesites by rugged compact instrumentation.

Another novel application developed with NIRDsfor the detection of Ca 2C was reported by Akkayaand Turkyilmaz. 94 They developed a squaraine-based

uorescentsensor forcalcium[Scheme 6, (47) ]. The probedevelopedwasverysensitivetoCa 2C concentrations in the


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4 8 12 16 20 24 28 32350

370

390

410

430

450

F l u o r e s c e n c e

i n t e n s i t y

( a . u . )

Concentration of Li (10 9 M)

Figure 17 Calibration curve for detection of Li C with

JCM-15C5 (45) .

NO

encyclopedia of analytical chemistry[1]

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