Integrated Electrochemical Microsystems for Genetic Detection of Pathogens at the Point of Care

Abstract

The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.

DNA‐Origami‐Driven Lithography for Patterning on Gold Surfaces with Sub‐10 nm Resolution

“…[14] Future studies could lead to the integration of this methodology within multiplexed microfluidic [11] and more multipurpose read out systems, [10a] For example, the integration of modular addressability with biological processes can be utilized for the high throughput analysis of biochemical reactions and biomolecular interactions that require control over proximity and special distribution. Thereby, the DNA origami stamp method presented here brings the opportunity for a more versatile and robust functionalization and patterning of surfaces for the creation of metamaterials [12a] with applications in nanoelectronics [7] and photonics [2c] .…”

“…[2c, 10] Nevertheless, these approaches have only used the DNA nanostructure to hold the chemical species on the surface and, to the best of our knowledge, have never been utilized to immobilize nucleic acids patterns on surfaces with sub-10 nm resolution providing an enable 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Submitted to 2 platform for potential applications such as multiplexed biochemical assays. [11] to the creation of metasurfaces [12] with potentially reconfigurable features. …”

DNA-Origami-Aided Lithography for Sub-10 Nanometer Pattern Printing

“…Finally, our computational model was utilized to simulate formation of more complex patterns (Figure 2c,d) that could be used for future implementation of our method with tailored properties. Future studies could lead to the integration of this methodology within multiplexed microfluidic [6] and multipurpose read out systems [4], for the systematic analysis of biochemical assays. …”

Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

“…First, the LAMP primer sets described here may be used with a variety of several less-expensive platforms (with diverse types of detection mechanisms), which include real-time turbidimeters, microfluidic chips (e.g., Gene-Z), and electrochemical or ultrasonic sensors (25,(40)(41)(42)(43). Besides being cheaper, these platforms are more-accessible alternatives to qPCR thermal cyclers.…”