Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Enabling Multiplexed Electrochemical Detection of Biomarkers with High Sensitivity in Complex Biological Samples.

The ability to perform multiplexed detection of various biomarkers within complex biological fluids in a robust, rapid, sensitive, and cost-effective manner could transform clinical diagnostics and enable personalized healthcare. Electrochemical (EC) sensor technology has been explored as a way to address this challenge because it does not require optical instrumentation and it is readily compatible with both integrated circuit and microfluidic technologies; yet this approach has had little impact as a viable commercial bioanalytical tool to date. The most critical limitation hindering their clinical application is the fact that EC sensors undergo rapid biofouling when exposed to complex biological samples (e.g., blood, plasma, saliva, urine), leading to the loss of sensitivity and selectivity. Thus, to break through this barrier, we must solve this biofouling problem.In response to this challenge, our group has developed a rapid, robust, and low-cost nanocomposite-based antifouling coating for multiplexed EC sensors that enables unprecedented performance in terms of biomarker signal detection compared to reported literature. The bioinspired antifouling coating that we developed is a nanoporous composite that contains various conductive nanomaterials, including gold nanowires (AuNWs), carbon nanotubes (CNTs), or reduced graphene oxide nanoflakes (rGOx). Each study has progressively evolved this technology to provide increasing performance while simplifying process flow, reducing time, and decreasing cost. For example, after successfully developing a semipermeable nanocomposite coating containing AuNWs cross-linked to bovine serum albumin (BSA) using glutaraldehyde, we replaced the nanomaterials with reduced graphene oxide, reducing the cost by 100-fold while maintaining similar signal transduction and antifouling properties. We, subsequently, developed a localized heat-induced coating method that significantly improved the efficiency of the drop-casting coating process and occurs within the unprecedented time of <1 min (at least 3 orders of magnitude faster than state-of-the-art). Moreover, the resulting coated electrodes can be stored at room temperature for at least 5 months and still maintain full sensitivity and specificity. Importantly, this improved coating showed excellent antifouling activity against various biological fluids, including plasma, serum, whole blood, urine, and saliva.To enable affinity-based sensing of multiple biomarkers simultaneously, we have developed multiplexed EC sensors coated with the improved nanocomposite coating and then employed a sandwich enzyme-linked immunosorbent assay (ELISA) format for signal detection in which the substrate for the enzyme bound to the secondary antibody precipitates locally at the molecular binding site above the electrode surface. Using this improved EC sensor platform, we demonstrated ultrasensitive detection of a wide range of biomarkers from biological fluids, including clinical biomarkers, in both single and multiplex formats (N = 4) with assay times of 37 and 15 min when integrated with a microfluidic system. These biosensors developed demonstrate the vast potential of solving the biofouling problem, and how it can enable potential clinically important diagnostic applications. This Account reviews our antifouling surface chemistry and the multiplexed EC sensor-based biodetection method we developed and places it in context of the various innovative contributions that have been made by other researchers in this field. We are optimistic that future iterations of these systems will change the way diagnostic testing is done, and where it can be carried out, in the future.

Ultrarapid Method for Coating Electrochemical Sensors with Antifouling Conductive Nanomaterials Enables Highly Sensitive Multiplexed Detection in Whole Blood.

The commercialization of electrochemical (EC)-sensors for medical diagnostics is currently limited by their rapid fouling in biological fluids, and use of potential antifouling coatings is hindered by the complexity and cost of application methods. Here, a simple ultrafast (< 1 min) method is described for coating EC-sensors with cross-linked bovine serum albumin infused with conductive, pentaamine-functionalized, graphene particles that can be stored at room temperature for at least 20-weeks, which provides unprecedented sensitivity and selectivity for diagnostic applications. The antifouling coating is applied directly on-chip using rapid heating via simple dip-coating, which provides unprecedented high levels of electrode conductivity for up to 9-weeks in unprocessed biological samples. This method is leveraged to develop a multiplexed platform for detecting clinically relevant biomarkers including myocardial infarction and traumatic brain injury using only 15 µL of blood. Single-digit pg mL-1 sensitivity is obtained within minutes in unprocessed human plasma and whole blood, which is faster and at least 50 times more sensitive than traditional enzyme-linked immunosorbent assays, and the signal generated is stable enough to be measured after 1 week of storage. The multiplexed EC-sensor platform is validated by analyzing 22 patient samples and demonstrating excellent correlation with reported clinical values.

A lab-on-a-chip for the concurrent electrochemical detection of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in saliva and plasma.

Rapid, accurate and frequent detection of the RNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and of serological host antibodies to the virus would facilitate the determination of the immune status of individuals who have Coronavirus disease 2019 (COVID-19), were previously infected by the virus, or were vaccinated against the disease. Here we describe the development and application of a 3D-printed lab-on-a-chip that concurrently detects, via multiplexed electrochemical outputs and within 2 h, SARS-CoV-2 RNA in saliva as well as anti-SARS-CoV-2 immunoglobulins in saliva spiked with blood plasma. The device automatedly extracts, concentrates and amplifies SARS-CoV-2 RNA from unprocessed saliva, and integrates the Cas12a-based enzymatic detection of SARS-CoV-2 RNA via isothermal nucleic acid amplification with a sandwich-based enzyme-linked immunosorbent assay on electrodes functionalized with the Spike S1, nucleocapsid and receptor-binding-domain antigens of SARS-CoV-2. Inexpensive microfluidic electrochemical sensors for performing multiplexed diagnostics at the point of care may facilitate the widespread monitoring of COVID-19 infection and immunity.

Low-Cost Graphene-Based Digital Microfluidic System.

In this work, the laser-scribing technique was used as a low-cost, rapid and facile method for fabricating digital microfluidic (DMF) systems. Laser-scribed graphene (LSG) electrodes are directly synthesized on flexible substrates to pattern the DMF electrode arrays. This facilitates the DMF electrodes' fabrication process by eliminating many microfabrication steps. An electrowetting test was performed to investigate the effectiveness of the LSG DMF electrodes in changing the contact angles of droplets. Different DMF operations were successfully performed using the proposed LSG DMF chips in both open and closed DMF systems. The quality and output resolution were examined to assess the performance of such patterned electrodes in the DMF systems. To verify the efficacy of the LSG DMF chips, a one-step direct assay for the detection of Legionellapneumophila deoxyribonucleic acid (DNA) was performed on the chip without the need for any washing step. The high specificity in distinguishing a single-nucleotide mismatch was achieved by detecting target DNA concentrations as low as 1 nM. Our findings suggest that the proposed rapid and easy fabrication method for LSG DMF electrodes offers a great platform for low-cost and easily accessible point-of-care diagnostic devices.

Capillary microfluidics in microchannels: from microfluidic networks to capillaric circuits.

Microfluidics offer economy of reagents, rapid liquid delivery, and potential for automation of many reactions, but often require peripheral equipment for flow control. Capillary microfluidics can deliver liquids in a pre-programmed manner without peripheral equipment by exploiting surface tension effects encoded by the geometry and surface chemistry of a microchannel. Here, we review the history and progress of microchannel-based capillary microfluidics spanning over three decades. To both reflect recent experimental and conceptual progress, and distinguish from paper-based capillary microfluidics, we adopt the more recent terminology of capillaric circuits (CCs). We identify three distinct waves of development driven by microfabrication technologies starting with early implementations in industry using machining and lamination, followed by development in the context of micro total analysis systems (µTAS) and lab-on-a-chip devices using cleanroom microfabrication, and finally a third wave that arose with advances in rapid prototyping technologies. We discuss the basic physical laws governing capillary flow, deconstruct CCs into basic circuit elements including capillary pumps, stop valves, trigger valves, retention valves, and so on, and describe their operating principle and limitations. We discuss applications of CCs starting with the most common usage in automating liquid delivery steps for immunoassays, and highlight emerging applications such as DNA analysis. Finally, we highlight recent developments in rapid prototyping of CCs and the benefits offered including speed, low cost, and greater degrees of freedom in CC design. The combination of better analytical models and lower entry barriers (thanks to advances in rapid manufacturing) make CCs both a fertile research area and an increasingly capable technology for user-friendly and high-performance laboratory and diagnostic tests.