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.

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.

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.

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.

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.

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.

Temperature non-uniformity detection on dPCR chips and temperature sensor calibration.

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of µL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (T M). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured T M values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured T M as the parameter. Even when the actual T M of the PCR solution was constant, the measured T M value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known T M as a temperature sensor. Among the non-contact temperature measurement methods, the proposed T M-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on T M allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.

An image-to-answer algorithm for fully automated digital PCR image processing.

The digital polymerase chain reaction (dPCR) is an irreplaceable variant of PCR techniques due to its capacity for absolute quantification and detection of rare deoxyribonucleic acid (DNA) sequences in clinical samples. Image processing methods, including micro-chamber positioning and fluorescence analysis, determine the reliability of the dPCR results. However, typical methods demand high requirements for the chip structure, chip filling, and light intensity uniformity. This research developed an image-to-answer algorithm with single fluorescence image capture and known image-related error removal. We applied the Hough transform to identify partitions in the images of dPCR chips, the 2D Fourier transform to rotate the image, and the 3D projection transformation to locate and correct the positions of all partitions. We then calculated each partition's average fluorescence amplitudes and generated a 3D fluorescence intensity distribution map of the image. We subsequently corrected the fluorescence non-uniformity between partitions based on the map and achieved statistical results of partition fluorescence intensities. We validated the proposed algorithms using different contents of the target DNA. The proposed algorithm is independent of the dPCR chip structure damage and light intensity non-uniformity. It also provides a reliable alternative to analyze the results of chip-based dPCR systems.

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.

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel.

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

Multiplexed digital polymerase chain reaction as a powerful diagnostic tool.

The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.

PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis.

Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K·s-1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

PCR past, present and future.

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.

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.

Infinite Selectivity of Wet SiO2 Etching in Respect to Al.

We propose and demonstrate an unconventional method suitable for releasing microelectromechanical systems devices containing an Al layer by wet etching using SiO2 as a sacrificial layer. We used 48% HF solution in combination with 20% oleum to keep the HF solution water-free and thus to prevent attack of the Al layer, achieving an outstanding etch rate of thermally grown SiO2 of ≈1 µm·min-1. We also verified that this etching solution only minimally affected the Al layer, as the chip immersion for ≈9 min increased the Al layer sheet resistance by only ≈7.6%. The proposed etching method was performed in an ordinary fume hood in a polytetrafluorethylene beaker at elevated temperature of ≈70 °C using water bath on a hotplate. It allowed removal of the SiO2 sacrificial layer in the presence of Al without the necessity of handling highly toxic HF gas.