# A Low-Cost and Portable Smart Instrumentation for Detecting Colorectal Cancer Cells
## 2 Materials and Methods
The measured signal is then processed using the microcontroller. Figure 1: Fluorescence imaging: (**a**) working principle of a typical fluorescence microscope and (**b**) a three-dimensional illustration of the proposed fluorimeter. IRFP702 fluorophore was expressed in the colorectal cancer cells (HCT116) using the lentiviral transduction method to test the developed prototype. The gene encoding IRFP702 was cloned into the pHAGE plasmid backbone (Addgene, Watertown, MA, USA) to construct pHAGE-IRFP702. To prepare a lentivirusrus, \(8\times 10^{5}\) 293-F (Thermo Fisher Scientific, Waltham, MA, USA) cells were plated for 24 h in 90% Dulbecco's Modified Eagle Medium (DMEM) medium (Thermo Fisher Scientific). One microgram of pHAGE-IRFP702 was mixed with 0.5 \(\upmu\)g of pSPAX.2 (Addgene) and 0.5 \(\upmu\)g pMD2.G (Addgene) in 100 \(\upmu\)L of Opti-Eagles minimum essential media (Opti-MEM) (Thermo Fisher Scientific). Polyethylenimine (PEI) (Sigma-Aldrich, Saint Louis, MO, USA) was added to 100 \(\upmu\)L of Opti-MEM to a final concentration of 120 \(\upmu\)g/ml. The DNA and PEI mixtures were mixed together and incubated at room temperature for 20 min to prepare a transfection mix. The transfection reaction was added to the cells drop-wise and incubated at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). The virus was collected 72 h after the transfection. For the transduction, \(1.5\times 10^{5}\) HCT116 cells (American Type Culture Collection) were plated for 24 h in HyClone(tm) McCoy's 5A Media (Fisher Scientific, Pittsburgh, PA, USA). The next day, the media was removed and replaced with 500 \(\upmu\)L of 90% McCoy's 5A medium with 4 \(\upmu\)g/mL of polybrene (Sigma-Aldrich) and incubated at 37 \({}^{\circ}\)C for 10 min. Five \(\upmu\)L of the virus was added to the cells and then centrifuged at 1500\(\times\)\(g\) for one hour. The cells were incubated at 37 \({}^{\circ}\)C with 5% CO  
Figure 3: Dimension of custom-built sample chamber printed with 3D printer which houses detector circuitry (CR-10): (**a**) top view, (**b**) isometric view, and (**c**) 3-dimensional view (exploded). All dimensions in this figure are given in cm. Figure 2: Off-the-shelf commercial components used to construct the prototype: (**a**) 3D printed sample chamber, (**b**) longpass filter, (**c**) microcontroller (Arduino Uno), (**d**) flashlight, (**e**) photodiode, (**f**) LCD display, and (**g**) holder (images are not to scale). overnight. The next day, the media was replaced with 90% [PERSON]'s 5A medium. Five days later the cells were analyzed on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) to ensure infection. The cells that were positive for IRFP702 were sorted from the negative population on a MoFlo Astrios EQ Cell Sorter (Beckman Coulter). IRFP702 expression in the cells was confirmed via scanning and imaging in the 700 channel on the Odyssey CLx (LI-COR Biosciences, Lincoln, Nebraska, USA). For fluorescence analyses, the fluorescent cells and matching non-fluorescent cells (control) were seeded on coverslips in 6-well culture dishes and cultured overnight in McCoy's 5A Media. The commercial glass slides (Cat. # 12-552-3, Fisher Scientific, USA) and coverslips (Cat. # 12-540C, Fisher Scientific, USA) have standard dimensions of 75 mm \(\times\) 25 mm (length \(\times\) width) and 25 mm \(\times\) 25 mm (length \(\times\) width) respectively. An EVOS Cell Imaging System (Thermo Fisher Scientific) was used to follow the growth of the cells. The cells were then fixed using 1% paraformaldehyde(PFA) (Sigma-Aldrich) on a glass slide which was used to test the prototype.