The beginning of the COVID-19 pandemic demonstrated how few point-of-care diagnostic tools were available that could be safely and easily operated by healthcare workers with no laboratory training. The gold-standard test, and initially the only test, used RT-PCR with nasal pharyngeal swabs (NPS). Two issues quickly emerged: 1) RT-PCR required central laboratory processing leading to significant time delays and 2) NPS collection causes discomfort, is inappropriate for ongoing repeat sampling of individuals (e.g., frontline healthcare workers) and poses difficulty when obtaining samples from some sections of the population (e.g. some elderly and young children). The Sal6830™ platform is a fully self-contained, RT-PCR point-of-care device for detecting SARS-CoV-2 from saliva that takes less than thirty minutes to complete. In this study we tested the usability of the Sal6830™ platform by healthcare workers unfamiliar with the instrument at two community clinics: Care 4 U Community Health Center (Miami, Florida, USA) and St. Mary’s Health Wagon (Wise, Virginia, USA). Staff participated in three tests: 1) determining SARS-CoV-2 status from blinded positive and negative saliva samples, 2) a clinical study comparing SARS-CoV-2 detection with a comparator point-of-care technology from the same patient and 3) completing a survey designed to measure comfort and confidence using the Sal6830™ point-of-care device having received no training. Participants overwhelming found the Sal6830™ platform easy and intuitive to use, successfully called SARS-CoV-2 status of contrived, blinded samples and measured a 93.3% overall percent agreement when comparing patient samples across two point-of-care technologies.### Competing Interest StatementAll authors have completed the ICMJE uniform disclosure form at [www.icmje.org/coi_disclosure.pdf][1] and declare: all support for work presented in this manuscript was provided by MicroGEM International PLC, including all funding, study materials, medical writing, article processing charges, etc; AS, JAH, JC, JDC, and BR hold stock in MicroGEM International PLC; JS, JDC and BR have pending patent applications; JS receives royalties from sales through the University of Otago benefit sharing scheme; AS, JAH, JC, JDC, RO, JS and BR are salaried employees of MicroGEM International PLC; Sal6830 development was supported by New Zealand grant MBIE UOOX1703 and the US NIH RADx program (2020); no other relationships or activities that could appear to have influenced the submitted work.### Funding StatementThis study was funded by MicroGEM International PLC### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:IRB 1313475 of Care 4 U Community Health Center gave ethical approval for this work IRB 1315538 of St Mary's Health Wagon gave ethical approval for the work. The IRB tracking number for this study was 2021278I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors [1]: https://www.icmje.org/coi_disclosure.pdf

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400-base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18-fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 +/- 2 and 166.48 +/- 3 bases, respectively.

In laser-induced fluorescence (LIF) detection, optimal alignment is essential in maximizing the fluorescent signal and, hence, detection sensitivity. Micro-total analysis systems (μTAS) involving microchip electrophoresis (ME) are challenged with alignment of the optics to the separation channel each run due to the single-use nature. Furthermore, μTAS devices that are designed to operate autonomously and by non-experts face additional challenges in performing alignment with micrometer resolution without human intervention. As part of the development of a total DNA analysis system, we set out to develop an automated alignment (AA) method to locate a 50-by-50 μm separation channel on a freely rotating microfluidic device in the absence of a fluorescent dye, accomplished without additional hardware. We detail the innate fluorescent signature attainable from laser excitation and the optimization of the algorithm to achieve AA at 84.6% success rate from 26 microchips. This AA method was a key element in realizing complete automation of the DNA analysis process in order to advance our instrument to a technology readiness level of 7. This is the first description of an AA method for ME (and centrifugal ME) with the purpose of providing transparent technical details to bridge the gap from 'fully integrated' to 'fully automated' instruments for point-of-detection, sample in-answer-out use cases. Written in the context of a forensic application, the AA method is adaptable for a wide range of bioanalytical applications involving LIF detection.

In the last decade, the microfluidic community has witnessed an evolution in fabrication methodologies that deviate from using conventional glass and polymer-based materials. A leading example within this group is the print, cut and laminate (PCL) approach, which entails the laser cutting of microfluidic architecture into ink toner-laden polyester sheets, followed by the lamination of these layers for device assembly. Recent success when applying this method to human genetic fingerprinting has highlighted that it is now ripe for the refinements necessary to render it amenable to mass-manufacture. In this communication, we detail those modifications by identifying and implementing a suitable heat-sensitive adhesive (HSA) material to equip the devices with the durability and resilience required for commercialization and fieldwork. Importantly, this augmentation is achieved without sacrificing any of the characteristics which make the PCL approach attractive for prototyping. Exemplary HSA-devices performed DNA extraction, amplification and separation which, when combined, constitute the complete sequence necessary for human profiling and other DNA-based analyses.

Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis. Primer concentrations were adjusted specifically for microdevice amplification, resulting in well-balanced short tandem repeat (STR) profiles. Furthermore, studies were performed to push the limits of the DNA polymerase to achieve rapid, multiplexed PCR on various substrates, including transparent and black polyethylene terephthalate (Pe), and with two distinct adhesives, toner and heat sensitive adhesive (HSA). Rapid STR-based multiplexed PCR amplification is demonstrated in 15 min on a Pe microdevice using a custom-built system for fluid flow control and thermocycling for the full 10-plex, and in 10 min for a smaller multiplex consisting of six core CODIS loci plus Amelogenin with amplicons shorter than 200bp. Lastly, preliminary studies indicate the capability of this PCR microdevice platform to be integrated with both upstream DNA extraction, and downstream microchip electrophoresis. This, coupled to the use of reagents that are compatible with lyophilization (lyo-compatible) for PCR, represents the potential for a fully integrated rotationally-driven microdevice for complete forensic DNA analysis.

To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng μL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification.

Current conventional methods utilized for forensic DNA analysis are time consuming and labor-intensive requiring large and expensive equipment and instrumentation. While more portable Rapid DNA systems have been developed, introducing them to a working laboratory still necessitates a high cost of initiation followed by the recurrent cost of the devices. This has highlighted the need for an inexpensive, rapid and portable DNA analysis tool for human identification in a forensic setting. In order for an integrated DNA analysis system such as this to be realized, device operations must always be concluded by a rapid separation of short-tandem repeat (STR) DNA fragments. Contributing to this, we report the development of a unique, multi-level, centrifugal microdevice that can perform both reagent loading and DNA separation. The fabrication protocol was inspired by the print, cut and laminate (PCL) technique described previously by our group, and in accordance, offers a rapid and inexpensive option compared with existing approaches. The device comprises multiple polyester-toner fluidic layers, a cyclic olefin copolymer separation domain and integrated gold leaf electrodes. All materials are commercially-available and complement the PCL process in a way that permits fabrication of increasingly sought after single-use devices. All reagents, including a viscous sieving matrix, are loaded centrifugally, eliminating external pneumatic pumping, and the sample is separated in <5 minutes using an effective separation length of only 4 cm (reagent loading to completed separation, is <37 minutes). The protocol for gold leaf electrode manufacture yielded up to 30 electrodes for less than $3 (cost of a 79 mm × 79 mm gold leaf sheet) and when using a device combining these electrodes and centrifugal reagent/polymer loading, the electrophoretic separation of STR fragments with two base resolution was demonstrated. This exemplary performance makes the device an ideal candidate for further integration and development of an inexpensive, portable and rapid forensic human identification system.

We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for genetic analysis of short tandem repeats (STR) via PCR amplification.

A fully integrated microfluidic chip for human identification by short tandem repeat (STR) analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-resolution separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (Leff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional instruments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total analysis system for point-of-collection human identification based on genetic analysis.

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

Forensic DNA analysis, conventionally, is time-consuming and laborious, requiring 8-10 hours for analysis. This lengthy processing time and the increased demand for forensic DNA analysis, has significantly contributed to the increasing backlog of forensic casework samples. Therefore, the demand for new analytical techniques that can reduce time and throughput has increased substantially. Typically, conventional STR analysis requires extraction and quantitation of the genomic DNA, multiplexed PCR amplification of the STR loci, and electrophoretic separation of the amplified STR fragments. Currently, separation of STR PCR product requires 30 minutes or more to complete and is performed on a large capillary electrophoresis instrument. By reducing the time requirements for analysis, the throughput of a crime laboratory can be increased.

In order to effectively reduce the analysis time required for conventional processing of forensic biological samples, the most time-consuming analysis step, PCR amplification, must be expedited. The transition from conventional to microfluidic PCR amplification allows for substantial (5-fold) time reduction. This work demonstrates the first use of an IR laser source for IR-mediated heating combined with non-contact temperature sensing for PCR amplification of STR loci for forensic DNA analysis on a microdevice. A conventional 3h 30 min amplification time is reduced to as little as 36 min in the microfluidic regime without sacrificing quality standards set by the forensic community.

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.

We demonstrate the use of an acid-labile surfactant (ALS) as a replacement for SDS for size-based protein separations in a microfluidic device. ALS is of interest to the proteomic field as it degrades at low pH and hence can be removed to reduce surfactant interference with down-stream MS. A range of SDS and ALS concentrations were tested as denaturants for microchip electrophoresis to investigate their effects on the separation of proteins from 18 to 116 kDa and to provide a suitable comparison between the two surfactants. The electrophoretic mobilities of the proteins were not significantly affected by the use of ALS instead of SDS. Protein separations with ALS are performed in less than 3 min, which is a significant decrease in the time compared with the previous ALS separations on a slab gel format. We also demonstrate the use of poly-N-hydroxyethylacrylamide as a dynamic, hydrophilic chip channel coating that can be applied with a rapid and simple protocol for size-based protein separation. The results reported here could significantly decrease the time and increase the attainable level of automation and integration of the front-end protein fractionation required for "top-down" proteomics.

In recent years, there has been an increasing demand for a wide range of DNA separations that require the development of materials to meet the needs of high resolution and high throughput. Here, we demonstrate the use of thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for DNA separations on a microfluidic chip. The viscosities of the N-alkoxyalkylacrylamide polymers are more than an order of magnitude lower than that of a linear polyacrylamide (LPA) of corresponding molecular weight, allowing rapid loading of the microchip. At 25 degrees C, N-alkoxyalkylacrylamide. polymers can provide improved DNA separations compared with LPA in terms of reduced separation time and increased separation efficiency, particularly for the larger DNA fragments. The improved separation efficiency in N-alkoxyalkylacrylamide polymers is attributed to the peak widths increasing only slightly with DNA fragment size, while the peak widths increase appreciably above 150 bp using an LPA matrix. Upon elevating the temperature to 50 degrees C, the increase in viscosity of the N-alkoxyalkylacrylamide solutions is dependent upon their overall degree of hydrophobicity. The most hydrophobic polymers exhibit a lower critical solution temperature below 50 degrees C, undergoing a coil-to-globule transition followed by chain aggregation. DNA separation efficiency at 50 degrees C therefore decreases significantly with increasing hydrophobic character of the polymers, and no separations were possible with solutions with a lower critical solution temperature below 50 degrees C. The work reported here demonstrates the potential for this class of polymers to be used for applications such as PCR product and RFLP sizing, and provides insight into the effect of polymer hydrophobicity on DNA separations.