Supplementary Information Paper-based microchip electrophoresis for point-of-care hemoglobin testing

Muhammad Noman Hasan1, Arwa Fraiwan1, Ran An1, Yunus Alapan1,2, Ryan Ung1, Asya Akkus1, Julia Z. Xu3, Amy J. Rezac4, Nicholas J. Kocmich4, Melissa S. Creary5, Tolulope Oginni6, Grace Mfon Olanipekun7, Fatimah Hassan-Hanga8, Binta W. Jibir9, Safiya Gambo10, Anil K. Verma11, Praveen K. Bharti11, Suchada Riolueang12, Takdanai Ngimhung12, Thidarat Suksangpleng12, Priyaleela Thota13, Greg Werner14, Rajasubramaniam Shanmugam11, Aparup Das11, Vip Viprakasit12,15, Connie M. Piccone16,17, Jane A. Little18,19,20, Stephen K. Obaro4,7, Umut A. Gurkan1,21*


List of Supplementary Videos
Video S1. Real-time tracking of hemoglobin bands in HemeChip during electrophoresis process Video S2.
Step-by-step HemeChip test procedure and analysis of results

Injection mold design
The HemeChip prototype design ( Fig. S1A&B) has been transformed into an injection moldable design ( Fig. S1C&D) with a plastic bottom and a top part (Fig. S2A&B) using a 1+1 mold. A 1+1 mold design is economic as it reduces the product cost due to less machine run time with reduced labor cost to produce each part. This is a very crucial aspect for mass producing a point-of-care (POC) single use cartridge as the cost per unit needs to be as low as possible. Optix® CA-41 Polymethyl Methacrylate Acrylic (PMMA) material was used in injection molding. The visual clarity of the Optix CA-41 is excellent. However, this visual clarity may be greatly impaired after the injection molding process due to surface finish of the mold ( Fig. S3A-C). The visual clarity of the HemeChip is crucial, since the detection method is based on image acquisition and analysis, and any impairment of visual clarity will significantly impact the performance of the detection system. Visual clarity and light transmission of the injection molded parts significantly improved after the mold underwent aluminum oxide polishing, which improved the visual clarity of the finished HemeChip part to its desired level (Fig. S3B&C). Optical transmission for HemeChip components with both standard machine finishing and aluminum oxide finishing were tested and compared (Fig. S3B). Co., Inc., Lincoln, NE). The optical transmission was measured at an angle of 0 for wavelengths ranging from 300 ~ 1000 nm. The results showed that the HemeChip components produced in the aluminum oxide polished mold have a much higher optical transmission (up to 80%) compared to the standard machine polished mold. Optical clarity and light transmission significantly improved after the mold underwent aluminum oxide polishing (Fig. S3B&C). HemeChip Reader consists of three major parts: (A) a rechargeable power supply, (B) a data acquisition system that collects current and voltage data for the duration of the test, and (C) an imaging system that records video and images for the duration of the test, which are transferred into an image processing software for analysis.

Micro-applicator design and operation
We designed a capillary-based micro-applicator to apply blood samples into HemeChip cartridge (Fig. S7).
This simple, easy-to-use component ensures a controlled and repeatable application of whole blood sample, and facilitates repeatable and reliable test results. The micro-applicator consists of a metal lancet and a PMMA sheet attached using double sided adhesive (DSA) (Fig. S7A). The spacing between the metal and the PMMA sheet is 150 µm. When the micro-applicator is dipped into the blood sample, it loads and retains a specific amount of the sample (Fig. S7B). A rectangular opening micro-machined onto the PMMA part of the micro-applicator (Fig. S7B) ensures this controlled amount of sample loading. The sample loading ports, located at the bottom of the HemeChip (Fig. S7C), are designed to provide just enough space to insert the micro-applicator (Fig. S7D), thus ensuring vertical alignment of the micro-applicator during sample application process. This design improves the accuracy and consistency of the application of blood samples at the same spot (Fig. S7E).

HemeChip test kit
The consumables and accessories needed for the sample preparation are as follows (Fig. S8): 1. A 20 µL capillary blood collection tube, this tube is used to collect exactly 20 µL of blood needed for the test, from either a heel or finger prick. The resulting lysing solution to blood ratio is 2:1.
3. A custom made applicator, which is dipped in the lysed blood mixture and applied to the surface of the cellulose acetate strip inside the HemeChip.

HemeChip Supplementary Materials
Analyst Page 14 of 35 4. 50 µL, and 200 µL fixed volume pipettes are provided to minimize user error. The 50 µL pipette is used to wet the cellulose acetate strip in the first step of the HemeChip preparation, and the 200 µL is used to load the buffer into the buffer ports just prior to starting the test.

5.
A battery-operated mini vortexer that is lightweight and portable, and is powered by four AA batteries.

Blood collection and HemeChip test protocol
1. Pipette 50 µL of the buffer solution onto the paper through the sample loading port, located at the bottom of the HemeChip cartridge. Then allow the paper to soak (Fig. S9A).
2. After administering a finger/ heel prick, touch the drop of blood with the capillary sample collection tube at a slight angle. Allow the tube to fill to the black line (Fig. S9B).

HemeChip Supplementary Materials
Analyst Page 15 of 35 3. Squeeze the top part of the tube to empty the blood into the tube containing lysing solution (Fig. S9C). 4. Place the tube on top of the vortexer to mix the blood and lysing solution for 20 seconds (Fig. S9D). 5. Invert the tube containing the blood mixture. Tap the tube on a solid surface to allow the mixture to reach the cap. Next, keep the tube inverted and open the cap. Dip the tip of the applicator into the blood mixture in the cap (Fig. S9E).
6. Stamp the blood mixture in the applicator onto the paper through the sample loading port by gently touching the paper surface with the applicator, while making sure not to puncture the paper with the applicator (Fig. S9F).