(E-182) A Novel Calibration and Prototyping Method for Point-of-Care Biomarker Assays

Introduction:: Colorimetry is widely used in assay systems due to its low-cost, ease-of-use, rapidity, moderate storage requirements, and intuitive visibility. However, the application is limited due to its relatively low sensitivity. One of the key issues is that conventional calibration methods with a fixed incubation time limit the detection range and detection time.

With expansion of the health market demand, there is an imminent shift from centralized laboratory assays, outpatient visits, and reactive medicine to decentralized, home-based precision medicine. Hence, there is an increased need for low-cost, accurate point-of-care devices for biomarkers collection, especially for underserved populations. For the detection of blood markers, the most common and rapid method is based on 3D printed soft lithography of microfluidic platforms, often created using a combination of glass and polydimethylsiloxane (PDMS). But this is usually limited to lateral flow. Others use resin to directly print devices, which is difficult to generate internal features and modify internal surfaces.

Materials and Methods:: Materials

Standard polymer resin, Sylgard 184, isopropyl alcohol, Deionized water (18.0 MU), Oxygen, (3-Aminopropyl)triethoxysilane, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, mouse C-Reactive protein (CRP) ELISA kit (ab222511), human cardiac troponin I (cTnI) ELISA kit (ab200016).

Method:

We first created a novel calibration surface with an optimal polynomic fitting function: Colorimetric Readout=f(Concentration, Time) as shown by the calibration mesh in Figure 1. a. When predicting the concentration, we check whether the predicted concentration value at the current time point is similar to the predicted concentration value at the previous time point. Once the predicted values converge within an accepted tolerance, we output the final predicted concentration. We compared the calibration mesh and conventional calibration curve for cardiac troponin I (cTnI) detection in mouse serum.

Second, we developed a PDMS on PDMS method to detect CRP based on soft lithography technique by generating sensing and top molds (Figure 1.b). and two corresponding PDMS layers. Then, we immobilized CRP antibodies in the sensing wells of the PDMS sensing layer. We combined the two layers of PDMS (Figure 1.c). Next we added 400 µL mixture of CRP test sample, buffer, and antibodies conjugated with horseradish peroxidase into the reaction chamber and incubated the platform on a microplate shaker with 350 RPM for 10mins. We then washed the reaction chamber by pumping 30mL washing. Finally, we added 5 µL TMB to the PDMS sensing wells by punching through the thin film (~0.5mm) on the top PDMS layer. Next, we captured images with CCD camera for colorimetric analyses.


Results, Conclusions, and Discussions:: Results:

The calibration mesh provided an output as early as 7 mins after adding the TMB substrate for high-concentration values. PDMS on PDMS fluidic device successfully detected the presence CRP as shown in the third well of Figure 1.d with a hue value of 0.27 as Figure 1.e. The other wells contained either no capture antibodies or antibodies not specific to CRP.

Conclusion:

Our novel calibration method demonstrated a dynamic and early output, extended the detection range, and increased system robustness compared to traditional calibration methods. Our PDMS on PDMS device provided a platform for CRP detection.

Discussion:

We also found PDMS adhered strongly to 3D printed resin layers. The combination of PDMS and resin layers can be valuable for future prototyping needs as the resin layer can be designed with complex fluidic channels. Since this material is durable, it can be easily assembled with inlet and outlet ports, and may allow for reuse.


Acknowledgements (Optional): : Thank you for NSF and PATHS-UP's support!

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