Microfluidic devices currently play an important role in many biological, chemical, and engineering applications, and there are many ways to fabricate the necessary channel and feature dimensions. In this review, we provide an overview of microfabrication techniques that are relevant to both research and commercial use. A special emphasis on both the most practical and the recently developed methods for microfluidic device fabrication is applied, and it leads us to specifically address laminate, molding, 3D printing, and high resolution nanofabrication techniques. The methods are compared for their relative costs and benefits, with special attention paid to the commercialization prospects of the various technologies.

In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\(^{8}\) guanine tags per secondary bead (\(7.5\times10^{6}\) biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)\(_{3}^{2+}\)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.

The COVID-19 pandemic has underscored the critical need for virus detection methods that are precise, simple, quick, and cost-effective. Electrochemical immunoassay-based methods are a practical solution given their ability to quickly, inexpensively, sensitively, and selectively detect the virus at the point of care. This study details the immunomagnetic capture of SARS-CoV-2 nucleocapsid protein in nasal samples, followed by electrochemical detection using gold nanoparticle labels on a screen-printed carbon electrode. We determined ideal conditions for the size of the gold nanoparticles and the length of the deposition time to maximize the electrochemical signal. The limit of detection for nucleocapsid protein was determined to be 2.64 ng/mL in PBS. The assay was successfully demonstrated to detect nucleocapsid protein in SARS-CoV-2-positive samples with a viral load as low as Ct = 25 (p-value < 0.0001 vs. negative patient control).

A schematic of the resistive-circuit method with three inlets for DI water, reagent and urine sample. The mixed fluids are transferred to a detection chamber.

Tuberculosis (TB) represents a major public health threat, with millions of new cases reported worldwide each year. A major hurdle to curtailing the spread of this disease is the need for low-cost, point-of-care (PoC) diagnostics. Mannose-capped lipoarabinomannan, a significant component of the Mycobacterium tuberculosis bacillus, has been heavily studied as a biomarker for TB, but with little success due to its complexation with endogenous components of body fluids in a manner that sterically interferes with its detection by ELISA and other immunoassays. Recent work by our group and others has shown that complexation can be disrupted with protein-denaturing protocols. By way of followup, we recently described an enzymatic digestion (Proteinase K) sample pretreatment that enables quantitative recovery of ManLAM spiked into healthy human control serum. Herein, we report on the transfer of our benchtop sample pretreatment methodology to an automated microfluidic platform. We show that this platform can be configured to: (1) carry out the pretreatment process with very little user interaction and, (2) yield recoveries for ManLAm spiked into control serum which are statistically indistinguishable from those achieved by the benchtop process. Plans to integrate this device with a portable sample reader as a possible basis for a PoC TB diagnostic system and analyze patient samples are briefly discussed.