Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond.

Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

IoT PCR for pandemic disease detection and its spread monitoring.

During infectious disease outbreaks, the centers for disease control need to monitor particular areas. Considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (POC) diagnostics, which could also create an Internet of Things (IoT) for healthcare via a global network. However, at present IoT based on a functional POC instrument is not available. Here we show a fast, user-friendly, and affordable IoT system based on a miniaturized polymerase chain reaction device. We demonstrated the system's capability by amplification of complementary deoxyribonucleic acid (cDNA) of the dengue fever virus. The resulting data were then automatically uploaded via a Bluetooth interface to an Android-based smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. The IoT system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by DNA or ribonucleic acid.

LAMP-on-a-chip: Revising microfluidic platforms for loop-mediated DNA amplification.

Nucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. Isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), exhibit characteristics ideal for point-of-care (POC) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. Other key advantages of LAMP are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. Polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making LAMP suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. In this review, we discuss the trends in miniaturized LAMP techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for POC applications alongside our opinion of the future development of miniaturized LAMP.

Nanostructured Gold Microelectrode Array for Ultrasensitive Detection of Heavy Metal Contamination.

Availability of potable water is a problem especially in developing countries as their water sources are often contaminated biologically as well as by heavy metals. Electrochemical methods are suitable for field application to monitor heavy metal contents. Noble metal electrodes with large surface areas are the subject of intensive development as they can yield an improved signal-to-noise ratio, enhanced sensitivity, and lower limits of detection (LOD). Here, we present a nanostructured Au microelectrode array subsequently modified by selective electrodeposition in lithographically defined circles within a partially cross-linked gelatin layer. This method increased its surface area by a factor of ≈1440 in comparison with an original lithography-based prepared array. The Au surface properties can be tailored by a degree of gelatin layer cross-linking. We used this array for an ultrasensitive detection of the As3+ ions content by stripping voltammetry achieving LOD of ≈0.0212 parts per billion (signal-to-noise ratio = 3.3), 470× below the content limit recommended by the World Health Organization for potable water. These nanostructured arrays were used to detect ions of other metals such as Cr, Cd, Hg, Cu, and Sb. In combination with a portable electrochemical device, we can envision an ultrasensitive heavy metal detection system for field application to monitor heavy metal contamination.

Portable Lock-in Amplifier-Based Electrochemical Method to Measure an Array of 64 Sensors for Point-of-Care Applications.

We present a portable lock-in amplifier-based electrochemical sensing system. The basic unit (cluster) consists of four electrochemical cells (EC), each containing one pseudoreference electrode (PRE) and one working electrode (WE). All four ECs are simultaneously interrogated, each at different frequencies, with square wave pulses superposed on a sawtooth signal for cyclic voltammetry (CV). Lock-in amplification provides independent read-out of four signals, with excellent noise suppression. We expanded a single cluster system into an array of 16 clusters by using electronic switches. The chip with an array of ECs was fabricated using planar technology with a gap between a WE and a PRE of ≈2 µm, which results in partial microelectrode-type behavior. The basic electrode characterization was performed with the model case using a ferricyanide-ferrocyanide redox couple (Fe2+/Fe3+) reaction, performing CV and differential pulse voltammetry (DPV). We then used this system to perform cyclic lock-in voltammetry (CLV) to measure concurrently responses of the four ECs. We repeated this method with all 64 ECs on the chip. The standard deviation of a peak oxidation and reduction current in a single channel consisting of 13 ECs was ≈7.46% and ≈5.6%, respectively. The four-EC configuration in each measured spot allows determination of nonperforming ECs and, thus, to eliminate potential false results. This system is built in a portable palm-size format suitable for point-of-care applications. It can perform either individual or multiple measurements of active compounds, such as biomarkers.

Palm-Sized Device for Point-of-Care Ebola Detection.

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics.

The Nanolithography Toolbox.

This article introduces in archival form the Nanolithography Toolbox, a platform-independent software package for scripted lithography pattern layout generation. The Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST) developed the Nanolithography Toolbox to help users of the CNST NanoFab design devices with complex curves and aggressive critical dimensions. Using parameterized shapes as building blocks, the Nanolithography Toolbox allows users to rapidly design and layout nanoscale devices of arbitrary complexity through scripting and programming. The Toolbox offers many parameterized shapes, including structure libraries for micro- and nanoelectromechanical systems (MEMS and NEMS) and nanophotonic devices. Furthermore, the Toolbox allows users to precisely define the number of vertices for each shape or create vectorized shapes using Bezier curves. Parameterized control allows users to design smooth curves with complex shapes. The Toolbox is applicable to a broad range of design tasks in the fabrication of microscale and nanoscale devices.

Microfluidic Superheating for Peptide Sequence Elucidation.

Herein, we introduce microfluidic superheating as a new method for peptide fragmentation prior to mass spectrometric analysis. The superheating conditions were found to be stable up to 240 °C for more than 30 min without elevated pressure or boiling of the aqueous sample. As proof of principle, we exposed the peptides ACTH1-10 and OVA257-264 to various superheating conditions, causing different degrees of decomposition. Optimized superheating conditions resulted in the entire peptide ladder sequence of the y-ions, allowing the amino acid sequence to be deduced from a single-stage mass spectrum. Thus, obtaining information in the same quality as from tandem mass spectrometry can be achieved by a single superheating step.

Catching bird flu in a droplet.

It is assumed that a timely mass administration of antiviral drugs, backed by quarantines and social distancing, could contain a nascent influenza epidemic at its source, provided that the first clusters of cases were localized within a short time. However, effective routine surveillance may be impossible in countries lacking basic public health resources. For a global containment strategy to be successful, low-cost, easy-to-use handheld units that permit decentralized testing would be vital. Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. In a sequential process, the viral RNA is isolated, purified, preconcentrated by 50,000% and subjected to ultrafast real-time RT-PCR. Compared to commercially available tests, the bioassay is equally sensitive and is 440% faster and 2,000-5,000% cheaper.

Correction: Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples.

Correction for 'Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples' by Sheng Ni et al., Lab Chip, 2023, 23, 1926-1934, https://doi.org/10.1039/D2LC01094A.

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing.

This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 µm to 5 µm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.

An integrated microfluidic platform for nucleic acid testing.

This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RT‒qPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28 min, including sample loading (5 min), ribonucleic acid (RNA) extraction (3 min), and RT-LAMP (20 min). The cost of each assay is ≈ $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform's versatility, allowing users to choose between RT‒qPCR or RT‒LAMP tests based on specific requirements.

Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples.

Differential scanning calorimetry (DSC) is a popular thermal analysis technique. The miniaturization of DSC on chip as thin-film DSC (tfDSC) has been pioneered for the analysis of ultrathin polymer films at temperature scan rates and sensitivities far superior to those attainable with DSC instruments. The adoption of tfDSC chips for the analysis of liquid samples is, however, confronted with various issues including sample evaporation due to the lack of sealed enclosures. Although the subsequent integration of enclosures has been demonstrated in various designs, rarely did those designs exceed the scan rates of DSC instruments mainly because of their bulky features and requirement for exterior heating. Here, we present a tfDSC chip featuring sub-nL thin-film enclosures integrated with resistance temperature detectors (RTDs) and heaters. The chip attains an unprecedented sensitivity of 11 V W-1 and a rapid time constant of 600 ms owing to its low-addenda design and residual heat conduction (∼6 µW K-1). We present results on the phase transition of common liquid crystals which we leverage to calibrate the RTDs and characterize the thermal lag with scan rates up to 900 °C min-1. We then present results on the heat denaturation of lysozyme at various pH values, concentrations, and scan rates. The chip can provide excess heat capacity peaks and enthalpy change steps without much alteration induced by the thermal lag at elevated scan rates up to 100 °C min-1, which is an order of magnitude faster than those of many chip counterparts.

Digital polymerase chain reaction duplexing method in a single fluorescence channel.

The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.

Smartphone integrated handheld (SPEED) digital polymerase chain reaction device.

We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

Improvement of the signal to noise ratio for fluorescent imaging in microfluidic chips.

Microfluidics systems can be fabricated in various ways using original silicon glass systems, with easy Si processing and surface modifications for subsequent applications such as cell seeding and their study. Fluorescent imaging of cells became a standard technique for the investigation of cell behavior. Unfortunately, high sensitivity fluorescent imaging, e.g., using total internal reflection fluorescence (TIRF) microscopy, is problematic in these microfluidic systems because the uneven surfaces of the silicon channels' bottoms affect light penetration through the optical filters. In this work, we study the nature of the phenomenon, finding that the problem can be rectified by using a silicon-on-insulator (SOI) substrate, defining the channel depth by the thickness of the top Si layer, and halting the etching at the buried SiO2 layer. Then the fluorescent background signal drops by = 5 times, corresponding to the limit of detection drop from = 0.05 mM to = 50 nM of fluorescein. We demonstrate the importance of a flat surface using TIRF-based single-molecule detection, improving the signal to a noise ratio more than 18 times compared to a conventional Si wafer. Overall, using very high-quality SOI substrates pays off, as it improves the fluorescence image quality due to the increase in signal-to-noise ratio. Concerning the cost of microfluidic device fabrication-design, mask fabrication, wafer processing, and device testing-the initial SOI wafer cost is marginal, and using it improves the system performance.