SERS-based microdevices for use as in vitro diagnostic biosensors.

Advances in surface-enhanced Raman scattering (SERS) detection have helped to overcome the limitations of traditional in vitro diagnostic methods, such as fluorescence and chemiluminescence, owing to its high sensitivity and multiplex detection capability. However, for the implementation of SERS detection technology in disease diagnosis, a SERS-based assay platform capable of analyzing clinical samples is essential. Moreover, infectious diseases like COVID-19 require the development of point-of-care (POC) diagnostic technologies that can rapidly and accurately determine infection status. As an effective assay platform, SERS-based bioassays utilize SERS nanotags labeled with protein or DNA receptors on Au or Ag nanoparticles, serving as highly sensitive optical probes. Additionally, a microdevice is necessary as an interface between the target biomolecules and SERS nanotags. This review aims to introduce various microdevices developed for SERS detection, available for POC diagnostics, including LFA strips, microfluidic chips, and microarray chips. Furthermore, the article presents research findings reported in the last 20 years for the SERS-based bioassay of various diseases, such as cancer, cardiovascular diseases, and infectious diseases. Finally, the prospects of SERS bioassays are discussed concerning the integration of SERS-based microdevices and portable Raman readers into POC systems, along with the utilization of artificial intelligence technology.

Advancing COVID-19 Diagnosis: Enhancement in SERS-PCR with 30-nm Au Nanoparticle-Internalized Nanodimpled Substrates.

Real-time polymerase chain reaction (RT-PCR) with fluorescence detection is the gold standard for diagnosing coronavirus disease 2019 (COVID-19) However, the fluorescence detection in RT-PCR requires multiple amplification steps when the initial deoxyribonucleic acid (DNA) concentration is low. Therefore, this study has developed a highly sensitive surface-enhanced Raman scattering-based PCR (SERS-PCR) assay platform using the gold nanoparticle (AuNP)-internalized gold nanodimpled substrate (AuNDS) plasmonic platform. By comparing different sizes of AuNPs, it is observed that using 30 nm AuNPs improves the detection limit by approximately ten times compared to 70 nm AuNPs. Finite-difference time-domain (FDTD) simulations show that multiple hotspots are formed between AuNPs and the cavity surface and between AuNPs when 30 nm AuNPs are internalized in the cavity, generating a strong electric field. With this 30 nm AuNPs-AuNDS SERS platform, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) can be detected in only six amplification cycles, significantly improving over the 25 cycles required for RT-PCR. These findings pave the way for an amplification-free molecular diagnostic system based on SERS.

Recent advances in point-of-care testing of COVID-19.

Advances in microfluidic device miniaturization and system integration contribute to the development of portable, handheld, and smartphone-compatible devices. These advancements in diagnostics have the potential to revolutionize the approach to detect and respond to future pandemics. Accordingly, herein, recent advances in point-of-care testing (POCT) of coronavirus disease 2019 (COVID-19) using various microdevices, including lateral flow assay strips, vertical flow assay strips, microfluidic channels, and paper-based microfluidic devices, are reviewed. However, visual determination of the diagnostic results using only microdevices leads to many false-negative results due to the limited detection sensitivities of these devices. Several POCT systems comprising microdevices integrated with portable optical readers have been developed to address this issue. Since the outbreak of COVID-19, effective POCT strategies for COVID-19 based on optical detection methods have been established. They can be categorized into fluorescence, surface-enhanced Raman scattering, surface plasmon resonance spectroscopy, and wearable sensing. We introduced next-generation pandemic sensing methods incorporating artificial intelligence that can be used to meet global health needs in the future. Additionally, we have discussed appropriate responses of various testing devices to emerging infectious diseases and prospective preventive measures for the post-pandemic era. We believe that this review will be helpful for preparing for future infectious disease outbreaks.

Highly Uniform Self-Assembly of Gold Nanoparticles by Butanol-Induced Dehydration and Its SERS Applications in SARS-CoV-2 Detection.

We report the development of a reproducible and highly sensitive surface-enhanced Raman scattering (SERS) substrate using a butanol-induced self-assembly of gold nanoparticles (AuNPs) and its application as a rapid diagnostic platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The butanol-induced self-assembly process was used to generate a uniform assembly of AuNPs, with multiple hotspots, to achieve high reproducibility. When an aqueous droplet containing AuNPs and target DNAs was dropped onto a butanol droplet, butanol-induced dehydration occurred, enriching the target DNAs around the AuNPs and increasing the loading density of the DNAs on the AuNP surface. The SERS substrate was evaluated by using Raman spectroscopy, which showed strong electromagnetic enhancement of the Raman signals. The substrate was then tested for the detection of SARS-CoV-2 using SERS, and a very low limit of detection (LoD) of 3.1 × 10-15 M was obtained. This provides sufficient sensitivity for the SARS-CoV-2 screening assay, and the diagnostic time is significantly reduced as no thermocycling steps are required. This study demonstrates a method for the butanol-induced self-assembly of AuNPs and its application as a highly sensitive and reproducible SERS substrate for the rapid detection of SARS-CoV-2. The results suggest the potential of this approach for developing rapid diagnostic platforms for other biomolecules and infectious diseases.

A strategy to enhance SERS detection sensitivity through the use of SiO2 beads in a 1536-well plate.

The development of rapid and accurate assays is crucial to prevent the rapid spread of highly contagious respiratory infections such as coronavirus (COVID-19). Here, we developed a surface-enhanced Raman scattering (SERS)-enzyme-linked immunosorbent assay (ELISA) method that allows for the screening of multiple patient samples with high sensitivity on a 1536-well plate. As the well number on the ELISA well plate increases from 96 to 1536, the throughput of the assay increases but the sensitivity decreases due to the low number of biomarkers and the increase in non-specific binding species. To address this problem, silica (SiO2) beads were used to increase the surface-to-volume ratio and the loading density of biomarkers, thereby enhancing sensitivity. Using a three-dimensional gold nanoparticle (AuNP)@SiO2 SERS assay platform on a 1536-well plate, an immunoassay for the nucleocapsid protein biomarker of SARS-CoV-2 was performed and the limit of detection (LoD) decreased from 273 to 7.83 PFU/mL compared to using a two-dimensional assay platform with AuNPs. The proposed AuNPs@SiO2 SERS immunoassay (SERS-IA) platform is expected to dramatically decrease the false-negative diagnostic rate of the currently used lateral flow assay (LFA) or ELISA by enabling the positive diagnosis of patients with low virus concentrations.

SERS-ELISA using silica-encapsulated Au core-satellite nanotags for sensitive detection of SARS-CoV-2.

The sensitive detection of viruses is key to preventing the spread of infectious diseases. In this study, we develop a silica-encapsulated Au core-satellite (CS@SiO2) nanotag, which produces a strong and reproducible surface-enhanced Raman scattering (SERS) signal. The combination of SERS from the CS@SiO2 nanotags with enzyme-linked immunosorbent assay (ELISA) achieves a highly sensitive detection of SARS-CoV-2. The CS@SiO2 nanotag is constructed by assembling 32 nm Au nanoparticles (AuNPs) on a 75 nm AuNP. Then the core-satellite particles are encapsulated with SiO2 for facile surface modification and stability. The SERS-ELISA technique using the CS@SiO2 nanotags provides a great sensitivity, yielding a detection limit of 8.81 PFU mL-1, which is 10 times better than conventional ELISA and 100 times better than lateral flow assay strip method. SERS-ELISA is applied to 30 SARS-CoV-2 clinical samples and achieved 100% and 55% sensitivities for 15 and 9 positive samples with cycle thresholds < 30 and > 30, respectively. This new CS@SiO2-SERS-ELISA method is an innovative technique that can significantly reduce the false-negative diagnostic rate for SARS-CoV-2 and thereby contribute to overcoming the current pandemic crisis.

Raman Thermometry Nanopipettes in Cancer Photothermal Therapy.

Raman thermometry based on surface-enhanced Raman scattering has been developed using nanopipettes in cancer cell photothermal therapy (PTT). Gold nanorods (AuNRs) are robustly epoxied on glass pipettes with a high surface coverage of ∼95% and less than 10 nm-wide nanogaps for intracellular thermometry and photothermal cancer therapy. The temperature changes could be estimated from the N≡C band shifts of 4-fluorophenyl isocyanide (FPNC)-adsorbed AuNRs on the Raman thermometry nanopipette (RTN) surfaces. An intracellular temperature change of ∼2.7 °C produced by altering the [Ca2+] in A431 cells was detected using the RTN in vitro, as checked from fura-2 acetoxymethyl ester (fura-2 AM) fluorescence images. For in vivo experiments, local temperature rises of ∼19.2 °C were observed in the mouse skin, whereas infrared camera images could not tract due to spatial resolution. In addition, a tumor growth suppression was observed in the PTT processes after an administration of the three AuNR-coated nanopipettes combined with a 671 nm laser irradiation for 5 min in 30 days. These results demonstrate not only the localized temperature sensing ability of FPNC-tagged AuNR nanopipettes in cell biology but also anti-cancer effects in photothermal cancer therapy.

SERS-based dual-mode DNA aptasensors for rapid classification of SARS-CoV-2 and influenza A/H1N1 infection.

We developed a dual-mode surface-enhanced Raman scattering (SERS)-based aptasensor that can accurately diagnose and distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 at the same time. Herein, DNA aptamers that selectively bind to SARS-CoV-2 and influenza A/H1N1 were immobilized together on Au nanopopcorn substrate. Raman reporters (Cy3 and RRX), attached to the terminal of DNA aptamers, could generate strong SERS signals in the nanogap of the Au nanopopcorn substrate. Additionally, the internal standard Raman reporter (4-MBA) was immobilized on the Au nanopopcorn substrate along with aptamer DNAs to reduce errors caused by changes in the measurement environment. When SARS-CoV-2 or influenza A virus approaches the Au nanopopcorn substrate, the corresponding DNA aptamer selectively detaches from the substrate due to the significant binding affinity between the corresponding DNA aptamer and the virus. As a result, the related SERS intensity decreases with increasing target virus concentration. Thus, it is possible to determine whether a suspected patient is infected with SARS-CoV-2 or influenza A using this SERS-based DNA aptasensor. Furthermore, this sensor enables a quantitative evaluation of the target virus concentration with high sensitivity without being affected by cross-reactivity. Therefore, this SERS-based diagnostic platform is considered a conceptually new diagnostic tool that rapidly discriminates against these two respiratory diseases to prevent their spread.

Sensitive and reproducible detection of SARS-CoV-2 using SERS-based microdroplet sensor.

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

Surface-Enhanced Raman Scattering-Based Dual-Flow Lateral Flow Assay Sensor for the Ultrasensitive Detection of the Thyroid-Stimulating Hormone.

The surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip has been considered a high-sensitivity sensor that can overcome the low sensitivity and the difficulty of quantitative analysis problems inherent in the colorimetric LFA sensor. In the SERS-based LFA strip reported so far, a liquid sample flows through the nitrocellulose membrane in a single pathway. In some cases, however, this single-flow approach still has a limitation in detection sensitivity. This study developed a conceptually new SERS-based dual-flow LFA sensor to improve the detection sensitivity in a single-flow LFA sensor. First, a 25 nm Raman reporter-labeled gold nanoparticle (AuNP) solution flowed through one way, and a 45 nm AuNP solution continuously flowed through another path. This sequential flow of two different AuNP solutions enables forming additional bright hot spots between 25 and 45 nm AuNPs in the test line, and the SERS signal is strongly enhanced. Using this SERS-based dual-flow LFA sensor, it was possible to detect thyroid-stimulating hormone less than 0.5 µIU/mL that cannot be measured with a SERS-based single-flow LFA sensor.

Recent advances in sensitive surface-enhanced Raman scattering-based lateral flow assay platforms for point-of-care diagnostics of infectious diseases.

This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.

Performance Evaluation of Surface-Enhanced Raman Scattering-Polymerase Chain Reaction Sensors for Future Use in Sensitive Genetic Assays.

We report a surface-enhanced Raman scattering (SERS)-based polymerase chain reaction (PCR) assay platform for the sensitive and rapid detection of a DNA marker (pagA) of Bacillus anthracis. Real-time quantitative PCR (RT-qPCR) has been recently considered a gold standard for the quantitative evaluation of a target gene, but it still suffers from the problem of a long thermocycling time. To address this issue, we developed a conceptually new SERS-PCR platform and evaluated its performance by sequentially measuring the Raman signals of B. anthracis DNA after the completion of different thermocycling numbers. According to our experimental data, SERS-PCR has lower limits of detection (LODs) than RT-qPCR under the small cycle number of 20. Particularly, it was impossible to detect a target DNA amplicon using RT-qPCR before the number of cycles reached 15, but SERS-PCR enabled DNA detection after only five cycles with an LOD value of 960 pM. In addition, the dynamic range for SERS-PCR (0.1-1000 pM) is wider than that for RT-qPCR (150-1000 pM) under the same condition. We believe that this SERS-PCR technique has a strong potential to be a powerful tool for the rapid and sensitive diagnosis of infectious diseases in the near future.

Analysis of deoxyribonuclease activity by conjugation-free fluorescence polarisation in sub-nanolitre droplets.

We report the analysis of deoxyribonuclease (DNase) activity by conjugation-free fluorescence polarisation in a droplet-based microfluidic chip. DNase is a DNA cleaving enzyme and its activity is important in the maintenance of normal cellular functions. Alterations in DNase activity have been implicated as the cause of various cancers and autoimmune diseases. To date, various methods for the analysis of DNase activity have been reported. However, they are not cost effective due to the requirement of large sample volumes and the need for the conjugation of fluorescent dyes. In this study, we have used ethidium bromide (EtBr), a DNA intercalating reagent, as a fluorescent reporter without any prior conjugation or modification of DNA. Degradation of DNA by DNase 1 was monitored at a steady state by making changes in the fluorescence polarisation of EtBr in droplets with a volume of 330 picolitre at a 40 hertz frequency under visible light. Using this technique, we successfully determined the half-maximal inhibitory concentration (IC50) of ethylenediaminetetraacetic acid (EDTA) for the inhibition of DNase 1 activity to be 1.56 ± 0.91 mM.

Quantitative Serodiagnosis of Scrub Typhus Using Surface-Enhanced Raman Scattering-Based Lateral Flow Assay Platforms.

A surface-enhanced Raman scattering-based lateral flow assay (SERS-LFA) technique has been developed for the rapid and accurate diagnosis of scrub typhus. Lateral flow kits for the detection of O. tsutsugamushi IgG (scrub typhus biomarker) were fabricated, and the calibration curve for various standard clinical sera concentrations were obtained by Raman measurements. The clinical sera titer values were determined by fitting the Raman data to the calibration curve. To assess the clinical feasibility of the proposed method, SERS-LFA assays were performed on 40 clinical samples. The results showed good agreement with those of the standard indirect immunofluorescence assay (IFA) method. SERS-LFA has many advantages over IFA including the less sample volume, simpler assay steps, shorter assay time, more systematic quantitative analysis, and longer assay lifetime. As SERS strips can be easily integrated with a miniaturized Raman spectrophotometer, field serodiagnosis is also more feasible.

SERS-based immunoassay using gold-patterned array chips for rapid and sensitive detection of dual cardiac biomarkers.

Cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) are important diagnostic biomarkers for acute myocardial infarction (AMI). Many efforts have been undertaken to develop highly sensitive detection methods for the quantitative analysis of these dual targets. However, current immunoassay methods are inadequate for accurate measurement of cTnI and CK-MB, due to their limited detection sensitivity. Thus, there is still an urgent demand for a new technique that will enable ultrahigh sensitive detection of these biomarkers. In this study, we developed a surface-enhanced Raman scattering (SERS)-based sandwich immunoassay platform for the ultrasensitive detection of cTnI and CK-MB. In this study, a monoclonal-antibody-immobilized gold-patterned chip was used as a SERS active template. Target samples and polyclonal-antibody-conjugated Au@Ag core-shell nanoparticles were then added. Using this SERS platform, the concentration of biomarkers could be quantified by monitoring the characteristic Raman peak intensity of Raman reporter molecules. Under optimized conditions, the limits of detection (LODs) were estimated to be 8.9 pg mL-1 and 9.7 pg mL-1 for cTnI and CK-MB, respectively. Thus, the proposed SERS-based immunoassay has great potential to be an effective diagnostic tool for the rapid and accurate detection of cTnI and CK-MB.

SERS-Based Immunoassays for the Detection of Botulinum Toxins A and B Using Magnetic Beads.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h.

Sensitive and Reproducible Immunoassay of Multiple Mycotoxins Using Surface-Enhanced Raman Scattering Mapping on 3D Plasmonic Nanopillar Arrays.

A surface-enhanced Raman scattering-based mapping technique is reported for the highly sensitive and reproducible analysis of multiple mycotoxins. Raman images of three mycotoxins, ochratoxin A (OTA), fumonisin B (FUMB), and aflatoxin B1 (AFB1) are obtained by rapidly scanning the surface-enhanced Raman scattering (SERS) nanotags-anchoring mycotoxins captured on a nanopillar plasmonic substrate. In this system, the decreased gap distance between nanopillars by their leaning effects as well as the multiple hot spots between SERS nanotags and nanopillars greatly enhances the coupling of local plasmonic fields. This strong enhancement effect makes it possible to perform a highly sensitive detection of multiple mycotoxins. In addition, the high uniformity of the densely packed nanopillar substrate minimizes the spot-to-spot fluctuations of the Raman peak intensity in the scanned area when Raman mapping is performed. Consequently, this makes it possible to gain a highly reproducible quantitative analysis of mycotoxins. The limit of detections (LODs) are determined to be 5.09, 5.11, and 6.07 pg mL-1 for OTA, FUMB, and AFB1, and these values are approximately two orders of magnitude more sensitive than those determined by the enzyme-linked immunosorbent assays. It is believed that this SERS-based mapping technique provides a facile tool for the sensitive and reproducible quantification of various biotarget molecules.

Fluoropolymer-Coated PDMS Microfluidic Devices for Application in Organic Synthesis.

In recent years there has been huge interest in the development of microfluidic reactors for the synthesis of small molecules and nanomaterials. Such reaction platforms represent a powerful and versatile alternative to traditional formats since they allow for the precise, controlled, and flexible management of reactive processes. To date, the majority of microfluidic reactors used in small-molecule synthesis have been manufactured using conventional lithographic techniques from materials such as glasses, ceramics, stainless steel, and silicon. Surprisingly, the fabrication of microfluidic devices from such rigid materials remains ill-defined, complex, and expensive. Accordingly, the microfluidic toolkit for chemical synthesis would significantly benefit from the development of solvent-resistant microfluidic devices that can be manufactured using soft-lithographic prototyping methods. Whilst significant advances in the development of solvent-resistant polymers have been made, only modest steps have been taken towards simplifying their use as microfluidic reactors. Herein, we emphasize the benefits of using a commercially available, amorphous perfluorinated polymer, CYTOP, as a coating with which to transform PDMS into a chemically inert material for use in organic synthesis applications. Its efficacy is demonstrated through the subsequent performance of photooxidation reactions and reactions under extremely acidic or basic conditions.

PEGylated nanographene-mediated metallic nanoparticle clusters for surface enhanced Raman scattering-based biosensing.

Surface-enhanced Raman scattering (SERS) is an optical spectroscopy technique that can detect a variety of analytes with high sensitivity and selectivity without any labels. Controlled clustering of metallic nanoparticles to prepare a new class of SERS nanotags is crucial for the ultra-sensitive detection of specific biological and chemical moieties because increased plasmonic hotspot junctions produce a greatly enhanced SERS signal. We report herein that controlled clustering of Au nanoparticles (AuNPs) was mediated by PEGylated nano-sized graphene (PNG) and that the PNG-induced AuNP clusters (PNG-AuNPCs) were highly sensitive SERS nanotags with colloidal stability for SERS-based biosensing. The AuNPs labeled with 4-mercaptopyridine as a Raman reporter were surface-modified with 1-aminomethylpyrene for the introduction of hydrophobic moieties, and were non-covalently complexed with PNG via π-π stacking and van der Waals forces. It resulted in the formation of PNG-AuNPCs that increased SERS intensity with an enhancement factor of 1.34 × 1011. The PNG induced a high degree of AuNP clustering by enhancing the non-covalent interactions between them, resulting in increased hotspot junctions at highly localized plasmonic centers. Furthermore, to show that the PNG-AuNPCs would serve as stable, reproducible, and highly sensitive SERS nanotags for biosensing, we formed sandwich-type immunocomplexes composed of the PNG-AuNPCs, immunoglobulin G (IgG) as the antigen, and magnetic beads. We found a linear relationship between SERS intensity and IgG concentration, with a limit of detection lower than 31.0 fM for IgG detection. Thus, the PNG-AuNPCs could be useful as SERS nanotags for highly sensitive SERS-based biosensing applications.

A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.