Freda Li, Department of Chemical and Biomolecular Engineering

Detection of Early Stage Prostate Cancer With Biomarker AMACR

Introduction Prostate cancer is the second most common type of cancer among men, but it can often by treated successfully when detected early. The current method of detection is known as Prostate Specific Antigen (PSA) blood testing. However, PSA testing is less than 65% specific and is limited in its ability to accurately detect prostate cancer in its early stages. Alpha-methylacyl-CoA rasemase (AMACR) is a unique protein biomarker for the assessment of prostate cancer. It is able to distinguish cancer cells from benign prostate cells with high sensitivity and specificity. This research involves the designing, fabricating and testing of a single use, disposable biosensor for the detection of AMACR in blood or urine. Recent clinical tests have shown that the detection of AMACR by our biosensor is 82.35% specific. AMACR has also show abilities to detect early stage prostate cancer.

Background This particular biosensor is designed and manufactured by a thick film screen printing technology. The substrate of the biosensor is polyethylene terephalate(PET) in a thickness of 0.18mm. The design of the biosensor has three-electrode configuration, working, counter and reference electrodes. This biosensor sensor detects the produced hydrogen peroxide (H2O2) which is generated from the enzymatic reaction of the substrate reactants, pristanic acid. The addition of AMACR will continue to

References 1.  Po-Yuan Lin et. al., Detection of Alpha-Methylacyl-CoA

Racemase(AMACR) a Biomarker of Prostate Cancer, in Patient Blood Samples using Nanoparticle Electrochemical Biosensors., Biosensors, 2013, 2,377-387.

2.  Llyod, M.D,et. Al, Alpha-Methyl-CoA Racemase an ‘obscure’ metabolic ensyme takes centre stage. FEBS. J. 2008, 275,1089-1102.

3.  Catalon W.J. et al, Prostate Cancer Detection in men with serum PSA concentrations of 2.6 to 4.0 ng/mL and benign prostate examination. Enhancement of specificity with free PSA measurements. JAMA 1997, 277, 1452-1455.

Acknowledgements Technical assistance provided by Professor C.C Liu, Ms. Laurie Dudik and Dr. Leo Kung of Electronics Design Center of Case Western Reserve University was gratefully accepted.

Conclusion Design and Methods The measurement of AMACR is carried out in two steps: 1. Prepare a substrate solution of pristanic acid and the involved enzymatic reaction components 2. Testing different concentrations of AMACR Preparing the substrate solution Pristanic acid, adenosine triphosphate(ATP), magnesium chloride(MgCl) and coenzyme A(CoA) is prepared in a phosophate buffer solution(PBS) at pH = 7. This substrate is then incubated at -20oC for 48 hours to facilitate the conversion of (2S)-pristanoyl-CoA to (2R)-pristanoyl-CoA. Testing for AMACR A mixture of substrate solution, ACOX3 and AMACR, total is placed onto the biosensor for testing.

Results

Amperometric IT measurements of this biosensor establishes a calibration curve relating the current output of the biosensor to AMACR concentration. The time point 350 seconds is chosen to relate the data for the system because it reaches steady state. A linear relationship between the current outputs and the AMACR concentrations is established as Y = 1.2*10-8X + 5.6*10-8

and R2=.9942 . These results demonstrate that a relatively inexpensive, yet effective biosensor for the detection of prostate cancer can be made using relatively small amounts of blood samples. PSA testing has shown that healthy men have AMACR levels below 0.4 ng/mL, however for men with prostate cancer PSA levels will increase above 0.4 ng/mL. Therefore, with a range of 0-0.4 ug/mL, early stage prostate cancer can be detected with this biosensor and can be treated successfully. In a recent clinical trial of 143 patients, this biosensor has shown to be 82.35% specific. This is very promising for an noninvasive, inexpensive biosensor.

Figure 3 shows a Differential Pulse Voltammetry test measuring current as voltage changes. This test follows Ohm’s Law, V=IR, where the slope of the graph shows the resistance on the sensor. It can be seen that as the concentration of AMACR increases the reaction requires more voltage input to make the reaction spontaneous. Also, the addition of AMACR to the substrate solution will transform (2R) to (2S) generating more H2O2 based on the quantity of AMACR present. Therefore, a higher concentration of AMACR will generate more H2O2 and a higher current output. The calibration curve (Figure 5) is made from the points at 350 seconds from amperometric IT testing. The time point 350 seconds is chosen because it is assumed that the solution is at steady state. At steady state, there should be a significant difference between each concentration of AMACR. A linear relationship Y = 1.2*10-8X + 5.6*10-8 with R2=.9942 is found and it shows a strong, linear relationship with AMACR and current output.

facilitate the production of of H2O2. Therefore, the measurement of H2O2 produced can be used to quantify the concentration of AMACR.