supplementary information for: microfluidics-based …...supplementary table 9. results of reference...

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Supplementary Information for:

Microfluidics-based diagnostics of infectious diseases in the developing world

Curtis D. Chin1, Tassaneewan Laksanasopin1, Yuk Kee Cheung1, David Steinmiller2, Vincent

Linder2, Hesam Parsa1, Jennifer Wang1, Hannah Moore1, Robert Rouse1, Gisele Umviligihozo3,

Etienne Karita3, Lambert Mwamarangwe4, Sarah Braunstein5, Janneke van de Wijgert4,6, Ruben

Sahabo5, Jessica Justman5, Wafaa El-Sadr5, Samuel K. Sia1†

1 Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, 1210

Amsterdam Avenue, New York, NY 10027

2 Claros Diagnostics, Inc., 4 Constitution Way, Suite E, Woburn, MA 01801

3 Rwanda Zambia HIV Research Group, Projet San Francisco, Kigali, Rwanda

4 Projet Ubuzima, Rue Akagera 716, PO Box 4560, Kigali, Rwanda.

5 Mailman School of Public Health, International Center for AIDS Care and Treatment

Programs (ICAP), Columbia University, 722 W 168th St # 14, New York, NY 10032

6 Academic Medical Center of the University of Amsterdam, Department of Internal Medicine,

Center for Poverty-related Communicable Diseases and Center for Infection and Immunity,

Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

† To whom correspondence should be addressed. Email: [email protected]

Nature Medicine doi:10.1038/nm.2408

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SUPPLEMENTARY FIGURES

Supplementary Figure 1. mChip assay preparation. a, Potential use of automated robots to spot

capture proteins on cassette. Alternatively, capture proteins can be spotted manually by

micropipette, as performed in this manuscript (procedure not shown). b, Close-up of liquid

dispensing machine. c, (Top) Image sequence demonstrating technique of loading small-diameter

plastic tubes with reagent plugs drawn from test tubes and separated by air gaps. Marks are

drawn on the tubing to indicate length (i.e. volume) of reagent plugs. Arrows (in white) indicate

reagent plugs, with three plugs positioned in series (far right). (Bottom) Manually loading


3

reagents in tubes using a 1 mL syringe, with arrows (in white) indicating direction of air and

fluid movement. d, Overall assay setup. Picture of cassette with a tube filled with sequence of

reagent plugs (here, colored dye) and syringe for generating vacuum. No other peripherals are

needed to run the mChip. Silver signals can be read by eye (similar to rapid tests), or with the

use of a sensitive absorbance reader, which can aid objective determination of positive and

negative results based on optical density.


4

Supplementary Figure 2. Compact reader for signal detection. a, Picture of compact reader.

b, Comparison of normalized absorbance values between reader and commercial scanner across

a range of samples.


5

Supplementary Figure 3. Modeling of silver development. a, Parameters for modeling silver

reduction. tOD,mid, a, ODmid, ODmin, ODmax and tn are determined from best-fit curves from

experimental data; fragAb and SAu,I are estimates based on literature. b, Silver enhancement of


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zone functionalized with 1:2 antibody to goat IgG antibody : BSA physisorption ratio. Data

points are mean absorbance values, and dashed line is best-fit curve (four parameter logistic

equation). Parameters are indicated accordingly. c, Dependence of tn on gold nanoparticle

density captured on the surface, with best-fit curve (exponential decay) as dashed line and best-

fit parameters listed in adjacent table. d, Experimental kinetic data of silver enhancement for

various antibody to goat IgG antibody : BSA physisorption ratio. Data points indicate mean

values, error bars indicate one s.d. Dashed lines are best-fit curves. e, Computational modeling

results (solid lines) superimposed with experimental data points. The difference (expressed as

normalized objective function) between the model and experiment is 9.2%.


7

Supplementary Figure 4. Selection of internal positive control. Two capture proteins, an

antibody to goat IgG antibody and a human IgG antibody, were compared as internal positive

controls. Dynamic range (i.e. difference in optical density between positive control and negative

control zones) and positive signal (i.e. optical density on positive control alone) were measured

in arbitrary units; shown are mean ± s.d.


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Supplementary Figure 5: Evaluation of HIV test in Rwanda on 101 total human samples from

Projet San Francisco with known hepatitis B and C status. a, Table listing number of truly HIV+

and HIV- samples which are positive or negative for HBV and HCV. The majority of samples

tested are positive for either HBV or HCV. b, Vertical scatter plot of mChip signal-to-cutoff

ratios for HIV+ and HIV- samples. c, ROC curve, with dashed line as random guess. d,

Contingency table and performance metrics (sensitivity and specificity) for HIV test.


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Supplementary Figure 6: Stability of silver reagents over time. Activity of silver solution A

varied between 93% and 104% of the original activity (at day = 0) for a direct assay using

physisorbed gold-labeled strepatavidin as the captured signal.


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Supplementary Figure 7: Stability of gold-labeled antibodies over time. Activity of gold-

labeled antibodies (specific for PSA) varied between 91% and 105% of original activity (at day =

4) for a model PSA assay on a 96-microwell plate.


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SUPPLEMENTARY TABLES

Supplementary Table 1. Survey results on demand for HIV-syphilis test in developing

countries.


12

Supplementary Table 2. Raw data collected at Columbia (HIV, sera or plasma). The detailed

reference results for specimen panel PRB204 are provided in Supplementary Table 9.


13


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Supplementary Table 3. Raw data collected at Columbia (syphilis, sera or plasma).


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Supplementary Table 4. Raw data for field trials at Muhima Hospital in Rwanda (HIV, whole

blood).


16


17

Supplementary Table 5: Raw data for field trials in Rwanda on samples collected from Projet

San Francisco (HIV, sera or plasma)


18


19

Supplementary Table 6. Raw data for field trials at Projet Ubuzima in Rwanda (HIV-syphilis,

sera or plasma).


20


21

Supplementary Table 7. Comparison of ELISA, rapid tests and mChip for HIV and syphilis.

Data for commercially available ELISA and rapid tests for HIV and syphilis are taken from

WHO evaluations18-22.


22

Supplementary Table 8. Social impact and cost-effectiveness of HIV-syphilis duplex POC test.


23

Supplementary Table 9. Results of reference tests provided by Seracare Life Sciences for HIV

panel PRB204


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SUPPLEMENTARY MOVIES

Supplementary Movie 1. Movie of HIV-syphilis duplex test (complete assay). Time lapse over

20 minutes (1200 s) for two duplex immunoassays, one with a sample which is negative for HIV

antibodies and positive for syphilis antibodies (top) and another with a sample which is positive

for HIV antibodies and negative for syphilis antibodies (bottom). Meandering zones are

functionalized with HIV antigen (left), syphilis antigen (middle), and antibody to goat IgG

antibody (right, positive control) as described in Supplementary Methods.


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Supplementary Movie 2. Movie of whole blood passing through microchannel. The mChip

can test whole blood samples without pre-processing or clogging of microchannels.


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SUPPLEMENTARY METHODS

Clinical need assessment via custom field surveys. We worked with the Venture Investment

Technical Assistance (VITA) group (in a grant supported by the Gates Foundation) to survey

over 60 health workers at antenatal clinics in India, Tanzania, and Rwanda. VITA conducted the

field surveys. This survey focused on antenatal care settings for a multiplexed HIV-STI

combination test because vertical transmission of STI’s from mother to child accounts for a large

burden of disease for STI’s. The results of this survey (Supplementary Table 1) showed strong

support for a multiplexed test that included HIV and syphilis. Rapid diagnosis would allow

treatment of the infant for HIV, and treatment of infected mothers for HIV and syphilis. Among

the Indian healthcare providers interviewed, all preferred rapid tests that simultaneously diagnose

HIV and STIs compared to HIV alone. Interviewees in Rwanda and Tanzania expressed strong

interest in HIV testing and dissatisfaction with current lab-based syphilis tests.

The VITA group (formed from Commons Capital and RTI) is composed of Richard

Snatcher, Doris Rouse, Meg Wirth, Elizabeth Bailey, and assessment study authors Christine

Poulos, David Rein, Jeffrey Petrusa, Ben Allayer, Anupa Bir, and Erin Hardy.

Social impact analysis. Using previously published methods developed by the Gates

Foundation and RAND1, the VITA group created quantitative models accounting for incremental

benefits (relative to current testing practices) such as avoided morbidity, avoided mortality,

avoided disability-adjusted life years (DALY), and avoided public treatment costs, as well as

incremental costs such as new testing costs. This analysis used country-specific disease


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prevalences, testing rates, number of pregnancies, sequelae from illness, and death from

sequelae. For syphilis, this model assumed an increased testing rate due to access of a

multiplexed STI test in remote regions, an increased proportion of tested patients who will

receive their results due to rapid turnaround time of testing, and more accurate diagnosis of

syphilis due to improved sensitivity and specificity. No incremental benefits of HIV testing were

assumed relative to current HIV rapid tests. The social impact analysis also makes conservative

estimates about market penetration over the course of 5 years of test administration (increasing

yearly from test introduction in year 2012) as well as availability of treatment.

The models showed significant health benefits predicted from screening and treating

syphilis-infected pregnant women (with a single dose of penicillin), such as avoidance of adverse

pregnancy-related outcomes (including congenital syphilis, low birth weight, neonatal death and

stillbirths) and adverse maternal health outcomes (i.e. the progression to tertiary syphilis)

(Supplementary Table 8). This analysis estimates that the deaths avoided from administration

of this test is over 5,000 in Rwanda, over 3,500 in Tanzania, and over 150,000 in India.

Importantly, the costs per DALY avoided ranged from $2 to $12, and were well below the per

capita GDP in each case (Supplementary Table 8); hence, this diagnostic test is cost-effective

according to WHO’s thresholds for evaluating cost-effectiveness of interventions (less than three

times GDP per capita for each DALY averted)2. In fact, the cost-effectiveness ratios of this test

are comparable to other key child health interventions such as immunizations and oral

rehydration therapy3.


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Visualization of microfluidic chip. “mChip” stands for mobile microfluidic chip for

immunoassay on protein markers. For SEM analysis (Fig. 1b), we diced the microfluidic chip

with a wire saw to produce a sample for observation of the cross-section of the channels. We

then cleaned the sample with isopropanol and DI water, prior to the evaporation of 15nm of gold.

We acquired SEM images with a Philips XL30 ESEM-FEG. For observation of the channel

meanders (Fig. 1c and 2a) we imaged the microfluidic chip under transmitted light using an

Olympus SZX10 stereoscope with Carl Zeiss Axiocam MRC (CCD) camera. We also used this

setup to acquire movies of the immunoassays (Supplementary Movies 1 and 2), and annotations

were made using ImageJ.

Surface modification. Like many ELISA assays using plastic 96-well plates, direct

physisorption of antigens worked well on our plastic microfluidic cassettes. (While we prepared

all cassettes used in this study by manually spotting antigens and capture proteins on the surface,

we have also employed robotic techniques for spotting; see Supplementary Fig. 1a,b for images

of liquid-handling robots). We selected gp41-gp36 envelope chimera for HIV (Biolink

International) and 17 kDa outer membrane protein for syphilis (Lee Labs), as these yielded the

best performance at Columbia. As an internal positive, we chose a secondary antibody to goat

IgG antibody (Invitrogen); when physisorbed, this protein provided even greater range of signal

(i.e. greater difference in OD relative to background signal on BSA-coated zone), larger signal

magnitude, and more reliable results than human IgG antibody (Supplementary Fig. 4).

Concentrations for physisorption were 2–4 µg/mL for HIV env, 15 µg/mL for TpN17, and 5

µg/mL for antibody to goat IgG antibody in bicarbonate buffer solution (with 10 µg/mL BSA

added to internal positive control to yield a 1:2 antibody to goat IgG antibody:BSA ratio).


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Vertical bars indicate mean values, with error bars representing one standard deviation (n=11

unique human samples).

After physisorption of capture protein for 1 hour in a humid chamber at room

temperature, we washed each meander zone three times with 1x phosphate buffer solution (PBS),

and then rinsed off the entire chip surface with deionized water (to remove any dirt or

particulates which may clog the microfluidic channels). We sealed the chips using a proprietary

adhesive as a backing, and incubated the channels for 45 min at room temperature with blocking

buffer (1% BSA in filtered PBS) (Sigma Aldrich) to prevent nonspecific protein adsorption, and

then cleared the channels. The cassettes were then either run immediately or stored in a dry

chamber at 4 degrees Celsius. We also blocked polyethylene tubing with 1-0.05% BSA-Tween

to reduce adsorption of sample and conjugate proteins.

Reagent loading. We loaded a series of reagents manually using a 1 mL syringe to draw

reagents into the PE tube (Supplementary Fig. 1c). We separate the reagent plugs with air

spacers each of 0.5 cm in length. For sera and plasma testing, the reagent sequence consists of

one lead wash buffer plug (0.3 cm long, ~ 1.3 µL), one plug of neat or diluted sample in

1%BSA/0.05% Tween-20 in filtered PBS (1.5 cm long, ~ 6.7 µL), four small plugs of washing

buffer (~ 1.3 µL each), one plug (2.5 cm long, ~ 11.2 µL) of gold nanoparticle-conjugated goat

antibody to human IgG antibody (1.45 µg/mL in 3% BSA/0.2% Tween-20 in filtered PBS), two

small plugs (~ 1.3 µL each) of washing buffer, and four small plugs (~ 1.3 µL each) of distilled

water. (Samples from Projet San Francisco were diluted 50x in 1%/0.05% BSA/Tween-20

buffer and plugs of 6 cm, ~26.8 µL, were loaded for each test). For testing with whole blood at


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Muhima Hospital, we metered neat sample (0.2 cm long, ~ 0.9 µL) not in the PE tubing but

rather in a short thin-walled polycarbonate (PC) tube which was connected to the pre-loaded PE

tube. We also added an extra wash buffer plug in between sample and gold conjugate plugs, and

eliminated the lead wash plug. In addition, the concentration and volume of gold-labeled goat

antibody to human IgG antibody was 0.4 µg/mL and 2.2 µL. We covered the tubes with

aluminum foil to minimize exposure to light and dust. Since silver nitrate can undergo

autoreduction from light exposure, we performed phostostability experiments of the silver (see

section titled “Stability of silver and gold-conjugated antibodies”).

While manual hand-loading of reagents is not ideal for large-scale implementation, we

note that robotic techniques can be employed to load these reagents with high precision and

throughput. The precision of manual loading does depend on skill of the user, but the use of

small-diameter tubing (0.86 mm) considerably reduces variability of plug volumes, which are

measured by marking lengths (Supplementary Fig. 1c). For instance, the case of variability of

± 1 mm plug length during tube loading would translate to a volumetric variability of ± 0.45 µL

(which, at a flowrate of 3 µL/min typical of buffer solutions, translates to ± 0.15 min or ± 9 s).

In practice, our precision in reagent loading is well below that range. We believe that

demonstration of hand-loading of regents is valuable because trained local workers in the

developing world can easily manufacture our device at a location and time close to when and

where the test will be conducted4.

Assay operation. We connected tubes to the inlet ports using a short PC tube connector, which

was used as a metering device for whole blood testing. To generate vacuum, we hooked up a 60


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mL syringe (connected with tubing) to the outlet port and pulled back the syringe a set distance

to generate a vacuum. While we used metal rods to hold the vacuum in each syringe, it would be

straightforward to use other cheap and locally available materials such as wood or plastic. The

magnitude of the vacuum was determined by relative displacement; up to 100 kPa (~1 atm) of

vacuum was possible using this method. We are also exploring the use of microfluidic cassettes

which have membranes (e.g. filter paper) for storing biohazardous waste on-chip.

With a vacuum pressure of 20 kPa (in the syringe), the residence times of the plugs were

2.5 min for sera and plasma samples (1 min for whole blood samples), 3.5 min for the gold-

labeled goat antibody to human IgG antibody, and 25 seconds per wash. After the final water

wash, we mixed silver solutions A and B (composed of silver nitrate and reducing agent, among

other chemicals) (supplied by Claros Diagnostics) and immediately loaded the silver into the

tubes preceded by a small wash of distilled water to avoid precipitation of AgCl. We ran silver

reagents continuously for 5 min (2.5 min for whole blood testing and 4.5 min for samples from

Projet San Francisco) and then immediately quenched the reaction with distilled water. The

overall assay time was 20 minutes or less. The silver signals can be observed by eye, or

quantified by using the compact reader as shown in Supplementary Figure 2a.

We estimate that the total failure rate is less than 10%. The data on the hundreds of

samples shown in this study were collected by five different co-authors over the course of weeks

with varying environmental conditions.


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Design of compact reader. We used two reader prototypes: one which can take measurements

of a single detection zone at once5, and another which can take simultaneous measurements of

four detection zones (shown in Supplementary Figure 2a). The dimensions are 5.5 x 5.5 x 4.5

inches and 6.5 x 6.5 x 5.5 inches, respectively (l x w x h). Both readers use the same light source

(super bright LED, 660 nm) and photodetector (photo sensitivity at 660 nm is 0.36 A/W)

(Hammamatsu), and have black Delrin plastic plates with 2 mm diameter pinholes (aligned

above each detection zone) to reduce noise from ambient light. The top plate is attached to linear

bearings for adjusting height when switching between cassettes. Both readers also use a custom-

designed microcontroller which is mounted underneath the bottom plastic plate and contains an

Atmel Mega32 microcontroller (for converting analog signals of transmitted light to digital

values, Digikey), among other components. Readings are taken upon a button push. The

transmittance values (i.e. mean values over a set of 32 rapid measurements over 1 s) are

displayed on an liquid crystal display (Digikey) and are logged manually; for Rwanda field trials,

we only used the reader shown in Supplementary Fig. 2a, and added the capability of

automatically logging the values and variance of transmittance in a laptop computer through

USB.

Data acquisition and processing. We calculated optical densities (OD) by recording the digital

intensity value (which is the converted analog output from the photodiode) both at maximum

transmission of light (a blank measurement, Io, of a filled meandering zone), and at transmission

of red light after silver development on the detection zone (I):

!

OD = "log II0

#

$ %

&

' (


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After collecting data, we set an OD threshold, and determined results to be positive

(when above the threshold) and negative (when below the threshold). In some cases, the OD

values were normalized against ODs in the internal positive control (functionalized with

antibody to goat IgG antibody). For testing at Columbia and Projet Ubuzima, the normalized

threshold value for HIV testing was 0.400 and 0.500, respectively, and for syphilis testing the

normalized threshold value was 0.220 at both locations. The threshold value for whole blood

HIV testing at Muhima Hospital was 0.024 and was 0.104 for sera and plasma testing on samples

from Projet San Franscisco. (Note: all figures in main text report normalized signal-to-cutoff

ratios, i.e. signals divided by threshold values). We also note that although hard threshold values

were used for purpose of evaluating sensitivity and specificity, in practice, ODs close to the

threshold values could be interpreted as an indeterminate result and would motivate additional

testing in a centralized lab.

Validation of compact reader. We compared measurements taken from reader with those taken

from a flatbed scanner (Hewlett Packard) as trends in normalized values should be similar

despite differences in light source (red vs. white light) and sensor position (detecting transmitted

vs. reflected light) (Supplementary Fig. 2b). We analyzed image intensity using ImageJ

(National Institutes of Health). A range of OD values (normalized with OD values on the

internal positive control) compared well between both modes of measurement.

Estimates of cost and time-to-fabrication of mChip. Because the reagent volume needed per

test is small, the cost of reagents per test is low. We estimate, given that 15 µL of HIV antigen at

concentration of 250x dilution (compared to stock solution) is needed for physisorption of an


34

HIV zone, the number of tests which can be performed with a stock antigen solution of 1 mL (at

1 mg/mL) is over 16,000. Since the antigen solution is priced at $210 per mg, the HIV antigen

cost per test is between $0.01 and $0.02. Using similar calculations, with a dilution factor of 33x

and a stock antigen solution of 2 mL (at 0.5 mg/mL) at $528/mg, the syphilis antigen cost per

test is $0.12. For antibodies to goat IgG antibody, with a dilution factor of 200x and a stock

antigen solution of 0.5 mL (at 2 mg/mL) at $80/mg, the estimated cost is between $0.01 and

$0.02. The gold-conjugated antibody to human IgG antibody, with 12 µL per test at dilution

factor of 80x, and a stock solution of 0.4 mL at $100/0.4mL, is estimated to be between $0.03

and $0.04. The silver solutions, blocking and wash buffers are estimated to be less than $0.01 in

total. Adding the costs of all reagents amounts to a total estimate of $0.20 per HIV-syphilis

duplex test. The cost of the compact reader is $74.50; we have previously developed a reader

which has one set of optics (instead of four), at a cost of $55.00.5

During the time for surface treatment of the cassettes (~1 hour of antigen coating, and 45

minutes for blocking), many cassettes can be treated in parallel. While we used manual pipetting

in this manuscript, we have employed robotic techniques for spotting antigens on cassettes in

parallel, which increases further the throughput of cassette preparation (Supplementary Figure

1a,b).

Stability of silver reagents and gold-conjugated antibodies. We investigated the long-term

stability of the silver solutions by loading silver solution A into microfluidics storage channels

with a cross-section of 350 um x 500um, made of identical materials as the mChip. We filled the

channels under normal light condition, sealed the access port with foil adhesive and then


35

packaged the microfluidics cassettes in foil pouches to mimic the envisioned packaging for the

product. During the filling process, the silver solution A was exposed to white light for

approximately 4 hours. The microfluidics cassettes were then stored at room temperature for up

to six months. At regular intervals, we extracted the silver solution A from the microfluidic

cassettes and used the stressed reagent in a model assay to assess the activity of the silver

solution A. The model assay was a direct assay performed by flowing a mixture of silver

solution A (stressed from the storage, or fresh reagent as a control) with fresh silver solution B in

a detection zone coated with gold-labeled strepatavidin. The results of the stability study show

that the activity of silver solution A, during the course of six months at room temperature, varied

between 93% and 104% of the original activity (at day = 0), indicating good stability of the

reagent in real-condition storage (Supplementary Fig. 6).

We also investigated the stability of the gold-labeled antibodies. For this purpose, we

studied the stability of monoclonal antibodies raised against prostate specific antigen (PSA),

which were labeled with gold colloids (gold-conjugated antibody to PSA). We diluted the gold-

labeled antibodies to PSA in a working buffer representative of a typical immunoassay

(phosphate buffer saline, containing 3% of bovine serum albumin, 0.2% Tween 20 and 0.09%

sodium azide) to a concentration representative of the working concentration in an immunoassay

(1.6 ug/mL). The diluted solutions were then stored at room temperature for up to seven months,

while the concentrated stock solutions of gold-labeled antibodies to PSA were maintained

refrigerated throughout the study as control material. We assessed the activity of the gold-

labeled antibodies to PSA using a model assay on a 96-microwell plate. We immobilized PSA

capture antibodies in the wells and incubated sequentially PSA samples, wash solutions, gold-


36

labeled antibodies to PSA, wash solutions and a mixture of silver enhancer. The results of the

stability study show that the activity of the gold-labeled antibodies to PSA solution, over the

course of seven months at room temperature, varied between 91% and 105% of the original

activity (at day = 4), indicating good stability of the reagent in real-condition storage

(Supplementary Fig. 7).

Modeling gold-silver amplification in mChip. We aimed to predict extent of silver formation

under a range of surface gold density and silver development time in order to minimize total

assay time. (We have previously modeled convection, diffusion, and surface adsorption in this

assay6; here, we focus on integrating the final step of the assay, silver development.) To guide

model development (in forming a general mechanism and determining best-fit values of kinetic

parameters e.g. rate constants), we first studied a simplified experimental system of physisorbing

different amounts of capture antibody to produce a range of surface gold density. (We controlled

the amount of antibodies to goat IgG antibody physisorbed to the surface by adding varying

amounts of BSA as a competitor). We used the procedure described previously to functionalize

one detection zone per channel with a particular ratio of antibodies to goat IgG antibody:BSA.

We loaded all tubes in a manner described previously with the following sequence of reagents:

one lead wash buffer plug (~ 1.3 µL), one plug (~ 11.2 µL) of gold nanoparticle-conjugated goat

antibodies to human IgG antibody, two small plugs (~ 1.3 µL each) of washing buffer, and four

small plugs (~ 1.3 µL each) of distilled water (i.e. a reagent sequence which lacked human

sample and trailing buffer washes). We ran all tests as described previously, but collected a

transmittance reading of the target zone every second during silver development to generate

absorbance curves over time.


37

The kinetics of silver reduction in mChip reveal a sigmoid-shaped response, with the

presence of an induction period followed by rapid growth of signal and termination of signal

growth (Supplementary Fig. 3d). For curve fitting, we used a variation of the four parameter

logistic equation,

!

OD = ODmin +ODmax "ODmin

1+ ea *(tOD,mid "t)

where OD, ODmin, ODmax are the optical density, minimum and maximum values respectively, t

is time of silver reduction and tOD,mid is the time at point of inflection, and a is a curvature

parameter (Supplementary Fig. 3b). (Curve fitting was performed using GraphPad Prism

software for nonlinear regressions; see Supplementary Fig. 6a for parameter values). R-squared

values for best-fit curves were 0.92 (antibody to goat IgG antibody only), 0.99 (1:1 antibody to

goat IgG antibody:BSA), 0.99 (1:2), 0.96 (1:4), 0.96 (1:8), and 0.71 (BSA only).

Together with the development of silver precipitates (established by AFM7,8 and SEM9 in

related systems), the silver reduction in mChip is believed to start similarly with the catalytic

formation of in situ silver nanoclusters around gold particles10,11 and undergo a general

mechanism involving a nucleation step followed by a autocatalytic surface-growth step12,13:

(The mechanism for the termination of signal growth may be due to non-linearity between

absorbance and silver at high silver density, or reduced silver deposition due to agglomeration of

nanoclusters to catalytically-inactive bulk metallic silver12,13.)


38

We focused on the first 5 minutes of silver reduction, during which sufficient signal is

generated and where nucleation and autocatalytic growth are prominent (Supplementary Fig.

3d, box outlined in red). We assume excess reducing agent (e.g. hydroquinone), irreversible

reactions, fast adsorption of reactants onto gold surface, even distribution of gold density across

the detection zone, and negligible silver precipitate desorption. Based on this reaction

mechanism we formed the following rate of silver formation:

!

ddt

[Ag(0)] = k1SAu [Ag(I)] + k2 [Ag(I)][Ag(0)]

with rate constant of nucleation (k1), rate constant of growth (k2), concentration ([]), and active

surface density of gold catalyst SAu (in units of moles of gold nanoparticles per square meter of

substrate surface). Due to attachment of silver precipitate around gold nanoparticles, the number

of active catalytic sites on the gold nanoparticles diminishes as the reaction progresses. We

therefore express SAu as a function of initial gold surface density bound (SAu,i) and the extent of

nucleation reaction, ξ

!

SAu = SAu,i 1"#( )

To estimate SAu,i, we assumed (1) equal rates of physisorption between antibody to goat

IgG antibody and BSA, (2) a surface density of 0.5 µg/cm2 of antibody to goat IgG antibody

(with molecular weight 150 kDa) for the antibody-only experimental condition14, (3) a 1:1

capture ratio of antibody to goat IgG antibody : gold-conjugated goat antibody to human IgG

antibody, (4) a 1:1 labeling ratio of gold nanoparticle : goat antibody to human IgG antibody

conjugate. Values of SAu,i for each experimental condition are given in Supplementary Fig. 6a.


39

To relate the amount of captured antibody with SAu,i, we used an average gold nanoparticle

diameter of 10 nm:

!

SAu,i = fragAb * 3.3x10"8

where fragAb is the percentage of antibody to goat IgG antibody (relative to total protein) in the

physisorption solution, and SAu,i is expressed.

For estimating ξ, we define a time tn, beyond which negligible silver is formed from

nucleation on gold nanoparticles (i.e. since all the surfaces of gold nanoparticles are already

covered by reduced silver), by finding the intersection between lines tangent to best-fit curve of

reduced silver formation at tOD,mid and t~0 (Supplementary Fig. 3b). We then express active

catalyst surface area as

!

SAu = SAu,i 1" ttn

#

$ %

&

' (

The dependence of tn on gold nanoparticle density captured on the surface is shown in

Supplementary Fig. 3c; we generalize the relationship with an exponential decay fit

!

tn = (tn,max " tn,min)e"k *SAu + tn,min

where tn, max , tn,min are tn at 100% and 0% of antibody to goat IgG antibody surface coverage and

k is a curvature parameter. R-squared value is 0.97.

We modeled the effect of flow parameters on the kinetic of reduction, by coupling the

convection/diffusion equation with the rate of silver formation at the boundary layer using weak-

form formulation (and assuming a parabolic velocity profile in the microchannel):

!

""t

[Ag(I)] +# $ (%DAg(I)#[Ag(I)] + [Ag(I)]u) = %ddt

[Ag(0)]


40

!

u = u0 1- y - 0.5h0.5h

"

# $

%

& '

2"

#

$ $

%

&

' '

with diffusion constant DAg(I) (1 x 10-10 m2-s-1), flow rate Q (5 x 10-11 m3-s-1), channel height h

(50 x 10-6 m), channel width w (100 x 10-6 m), velocity u and max velocity u0 (= 3*Q/(2*h*w)),

and initial concentration of Ag(I) (0.01 mol-m-3).

We then tuned the parameters by comparing the model output with the experimental data

for 5 different gold concentrations. Nucleation rate constant (k1) and autocatalytic rate constant

(k2) were determined according to minimization of the model error. There are several

optimization algorithms available to minimize the magnitude of an objective function (e.g. the

sum of the errors between the model output and the experimental data over time and over the

five different gold concentrations). We chose “pattern search algorithm” (Direct search toolbox,

Matlab) since it handles the constrained nonlinear optimization problems in a reasonable

timeframe and does not require the function to be differentiable and continuous. Minimizing the

objective function, we determined the nucleation rate constant (k1) and autocatalytic rate constant

(k2) to be 10-6 s-1 and 20 m3mol-1s-1 respectively. (We converted silver surface density to optical

density by assuming a linear relationship with scaling factor of

!

ODt=5min

[Ag(0)]t=5min

).

Supplementary Fig. 3e compares modeling results (after optimization of rate constants;

solid lines) with experimental results (filled circles; error bars indicate one standard deviation)

for silver enhancement over five minutes at different surface densities of captured gold

nanoparticles. To determine the goodness of fit between model and experiment, we normalized

the objective function by the total integral of the experimental curves over the five gold


41

concentrations. The difference (expressed as normalized objective function) between the model

and experiment is 9.2%.

Trials at Columbia University. We screened 12 HIV antigens, which were all recombinant

envelope proteins (e.g. gp41, gp120, and gp36), from various commercial sources and from

different strains (the “chimeric” antigen selected for this study contained epitopes from envelope

proteins on HIV type 1 M and O groups and on HIV type 2). We screened 8 treponema

(syphilis) antigens, most of which were recombinant outer membrane proteins (e.g. 15, 17, and

47 kDa antigens), from primarily commercial sources.

Fig. 2 and Supplementary Tables 2 and 3 report data from single-analyte testing

performed at Columbia University. For each assay we used 6.7 µL of sample from panels

purchased from Zeptometrix (catalog numbers CN6710 and K-ZMC002) and Seracare (catalog

numbers QHV711, PRB204, QSS701, and VRZ602), and performed each assay as described in

previous sections (“surface modification”, “reagent loading”, “assay operation”, and “data

acquisition and processing”). Samples used for evaluating HIV test performance had been

validated with commercial ELISA tests and/or rapid tests by expert third-party suppliers; in some

cases, additional testing with Western Blot was also provided by the supplier. We reported HIV

status based on vendor-provided results from reference test(s): positive (i.e. pos), negative (i.e.

neg), and indeterminate (for samples which had conflicting results from independently-

performed commercial reference tests). Nine samples from commercial specimens exhibited

conflicting reference results (Supplementary Table 9); since the true disease states of these

specimens are not clear, we did not include them in the analysis. For syphilis testing, we ran


42

positive samples that were previously independently validated with treponema-specific tests

(and, for several of these, the RPR status was also known); for syphilis negative samples, RPR

results were available and used as the reference standard.

Rwanda field trials. We ran whole blood samples (validated for HIV) at Muhima Hospital, a

district-level hospital in Kigali, Rwanda (Fig. 3 and Supplementary Table 4), as described in

previous sections. Sample collection and reference testing were performed by health workers in

Muhima Hospital and not by any of the authors. All HIV-positive samples were positive for a

series of rapid tests (First Response, Uni-Gold, and Capillus), and all HIV-negative samples were

only tested once (with First Response). As Muhima experiences regular frequency of HIV

testing for PMTCT and VCT counseling as well as for HIV/AIDS monitoring, it allowed testing

of human whole-blood samples that were recently collected via venipuncture (our ethics protocol

as approved by the Ministry of Health in Rwanda allowed testing of archived, validated

specimens). Over the span of three days, we tested 70 human samples, of which 42 were

positive and 28 were negative, based on availability from patients recently presenting to the

clinic, and selected to ensure roughly equal numbers of positives and negatives to increase

statistical confidence of sensitivity and specificity analysis. The HIV-negative blood samples

came from patients who were receiving antenatal care or undergoing voluntary, counseling and

testing. The HIV-positive blood samples came from patients who were getting tested for CD4+

cell counts and undergoing antiretroviral treatment (ARV). We tested whole-blood specimens

within several days after collection. Gender was not known for HIV-positive samples (most of

which were collected from patients getting CD4 counts); there were about equal numbers of

women and men for HIV-negative sample group. All human samples were coded.


43

A potential source of inaccurate test results for HIV is liver disease caused by viral

hepatitis15,16. We evaluated, in Rwanda, the HIV accuracy of mChip on samples from patients

who were also tested for hepatitis B and hepatitis C (Supplementary Fig. 5 and Table 5). These

archived samples were collected from Projet San Francisco, an international NGO registered in

Kigali, Rwanda, which provides HIV voluntary counseling and testing for couples, among other

health care-related activities. Sample collection and reference testing were performed by health

workers in Projet San Francisco and not by any of the authors. From a set of 101 samples, all but

one had tested positive for either (or both) HBV and HCV (Supplementary Fig. 5a); reference

testing for HIV was performed with a series of rapid tests (Abbott’s Determine, Trinity Biotech’s

Uni-gold, and Trinity Biotech’s Capillus) and confirmed with ELISA (Vironestika 4th generation

Ag/Ab), and reference testing for HBV and HCV was performed with ELISAs (Abbott Murex

HBsAg v3.0 and Abbott Murex anti-HCV v4.0, respectively). Despite the high prevalence of

viral hepatitis in this sample set, the sensitivity of mChip was high at 100%, and the specificity

was 94%, with four false positives (Supplementary Fig. 5b-d). The accuracy of HIV test on the

high-prevalence hepatitis-positive sample set was similar to the accuracy achieved at Columbia

(where most samples were known to be negative for HBV and HCV) and at Projet Ubuzima.

In addition we ran HIV-syphilis duplex tests at Projet Ubuzima, an international NGO

registered in Kigali, Rwanda (Fig. 4 and Supplementary Table 6). The archived specimens

were collected from high-risk women (most self-identified as sex worker) participating in an

HIV incidence study. In this separate study, 800 high-risk women were tested cross-sectionally

(24% tested HIV-positive), and 400 of them were subsequently followed in a cohort study for 12


44

to 24 months (with 19 HIV seroconversions). The women were tested for HIV at all study visits,

and for syphilis and other STIs at cohort enrollment, month 6 and month 12. For HIV, a series of

rapid tests were performed at Projet Ubuzima (Premier Medical Corp’s First Response, Trinity

Biotech’s Uni-gold, and Trinity Biotech’s Capillus). All HIV-positives by rapid test were also

independently confirmed by ELISA (Abbott AxSYM platform) by the Perinatal HIV Research

Unit in Johannesburg, South Africa. All HIV-negatives (by First Response rapid test) were also

pooled and independently tested by HIV-1 RNA PCR at the National Reference Laboratory in

Kigali, Rwanda; no HIV-1 RNA was detected. For syphilis, all women were tested at Projet

Ubuzima by a non-treponemal test, RPR (Human), and RPR-positives were confirmed by a

treponemal-specific test, TPHA (Human). For this study, all negative samples were also

confirmed by TPHA. For both RPR and TPHA, titers are expressed as the highest dilution factor

in which samples yield a positive result. All human samples were coded. There were no criteria

for selection other than specimen availability, which was the main constraint as these samples

were also used for other studies. Of a total 67 human samples tested, 66 were sera and one was

plasma.

Evaluation of test performance. We calculated sensitivity and specificity for the Columbia,

Muhima, San Francisco and Ubuzima datasets. Given the number of true positives (TP), true

negatives (TN), false positives (FP), and false negatives (FN), the performance metrics are:

!

sensitivity =TP

FN +TP

!

specificity =TN

FP +TN

95% confidence intervals are calculated using the formula:


45

!

p ±1.96 * p(1" p)n

where p is sensitivity (or specificity) expressed as a proportion and n is the number of samples

analyzed17. For cases where 100% sensitivity or specificity was reported, a p-value of 0.999 was

used. Receiver operating characteristic (ROC) curves were created using GraphPad Prism.

Special considerations for testing diagnostic devices in developing countries. Power,

environment, and transport of materials all need to be considered when running portable

immunoassays in Rwanda. To protect the electronics on the reader against sharp increases in

current (which we experienced on-the-field with fluctuations in ground electricity), we put in a

step-down adapter and a power regulator; alternatively, the compact reader could also be

operated on a single 9V battery for 660 runs continuously. The environment (specifically at

regional and rural clinics) can fluctuate widely in heat and humidity because of the lack of air

conditioning; see Supplementary Figures 6 and 7 for data on characterizing thermostability and

phostability of silver and gold-labeled antibody reagents. In addition, since windows are often

opened during hot days, dust can pose a problem (e.g. by disrupting microfluidic flow). It is

common to find instruments covered when not in operation to prevent damage from dust.

When conducting the development and evaluation of diagnostic tests in developing

countries, it is important that the study protocol undergo scientific and ethical review by

institutional and national guidelines17. Our study was reviewed and approved by the National

Ethics Review Committee in the Ministry of Health in Rwanda in 2007 and reapproved in 2009

(RNEC No. 164, “Columbia University Diagnostic Chip for Infectious Disease in Rwanda”).


46

Translation of POC diagnostic technologies to the developing world. Although a technology

may exhibit scientific and technological attributes attractive for use both in the U.S. and in the

developing world, the pathway to implement the technology in the field is vastly different in the

two settings. With the VITA group in a grant funded by the Gates Foundation, we developed a

detailed model on the challenges concerning regulatory approval, distribution, and financial

sustainability of our mChip device. The report concludes that based on an initial launch in India,

Rwanda, and Tanzania (using the price points acceptable to our end users, according to the

custom survey), such a point-of-care diagnostics device can achieve financial self-sustainability

within a few years.

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