CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.

Novel strategies for the simultaneous and portable detection of multiple analytes are highly favorable for clinical diagnosis and healthcare. Conventional colorimetric enzyme-linked immunosorbent assay (ELISA) is a widely used laboratory technique for medical diagnostics, quality control, and research applications. However, nonspecific absorption of proteins may lead to a reduction of functional sites, resulting in high background and low sensitivity in ELISA. Herein, we report a simple method of functionalization of poly(methyl methacrylate) (PMMA) with polylysine to be used as the microfluidic microplate substrate for enhanced ELISA, enabling rapid, ultrasensitive, and multiplexed detection of infectious diseases. FTIR and fluorescence microscopy characterization confirmed high amine densities on polylysine-modified PMMA surface, resulting in high detection sensitivity of the colorimetric ELISA on the PMMA microdevice. The ultrasensitive polylysine-modified microplate can immobilize protein within 20 min and results of the assay can be viewed by the naked eye or scanned through a simple desktop scanner for quantitative analysis within 90 min. A sandwich-type immunoassay for the rapid and sensitive detection of immunoglobulin G (IgG), hepatitis B surface antigen (HBsAg), and hepatitis B core antigen (HBcAg) was demonstrated as a proof-of-concept for multiplexed detection. The limits of detection (LOD) of 200.0 pg/mL for IgG, 180.0 pg/mL for HBsAg, and 300.0 pg/mL for HBcAg were achieved, without any specialized equipment like a microplate reader. The surface-modified microchip exhibited about 10-fold higher sensitivity than traditional microplates. This surface-modified microplate has tremendous potential as a point-of-care multiplexed testing platform for many applications ranging from clinical diagnosis to environmental monitoring, particularly in resource-limited settings.

In this study, we present a novel paper/polymer hybrid microfluidic Rotary Chip, integrated with aptamer-functionalized graphene oxide (GO) nanosensors, which enables not only efficient reagent delivery but also quantitative multiplexed detection. Rapid simultaneous detection of two major aflatoxins, aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), was successfully achieved to demonstrate the effectiveness of this hybrid rotary chip. Aflatoxins, as potent natural carcinogens, represent a significant global food safety challenge. Rapid and sensitive detection methods are crucial for effective monitoring. The RotaryChip design involves manually rotating the top poly(methyl methacrylate) (PMMA) plate over the bottom plate around a central screw, providing a simple strategy for rapid and efficient reagent delivery for multiplexed assays and high-throughput analysis without the need for sophisticated external pumps or pneumatic valves. The paper substrate in this hybrid microfluidic plate facilitates the facile integration of nanosensors in detection wells without the need for elaborate surface modification. The entire assay was completed in approximately 30 min and did not involve any washing steps. The platform achieved detection limits as low as 0.7 μg/kg for AFB1 and 0.5 μg/kg for AFB2. We further validated the platform by accurately detecting and quantifying AFB1 and AFB2 in spiked cooking oil samples with high specificity. Owing to its high simplicity and specificity, fast analysis, simple and efficient reagent delivery, and its multiplexing and quantitative capability, this paper/PMMA hybrid microfluidic nanosensing platform has tremendous potential for environmental and food safety surveillance, including multiplexed detection of different toxins and pathogens, particularly in low-resource settings.

Pressure-based signal readout is promising for developing instrument-free immunoassays, but new detection methods are desirable to further advance the applicability of point-of-care testing (POCT). Herein, we developed a syringe-hosted load-and-read immunoassay device using autoinjected distance readout in paper inserts. The device was composed of multiple disposable syringes as the host for immuno-recognition, a three-dimensional (3D)-printed loading magazine of the syringes, and paper inserts to the magazine for distance signal readout. Using prostate-specific antigen (PSA) as a model target, the immuno-recognition system was constructed on the inner cylinder walls of the syringes. The immuno-captured platinum nanoparticles (Pt NPs) catalyzed the decomposition of H2O2 to produce O2 in the cylinders, driving the automatic quantitative injection behavior of the syringes. By loading the syringes into the magazine, the autoinjected liquids were received by the paper inserts to display the immunoassay signals as a visual traveling distance of liquids. PSA was determined with a low limit of detection (LOD) of 0.32 ng/mL and high accuracy in testing clinical serum specimens. Given the advantages of the simple syringe-hosted immuno-recognition method and the load-and-read signal readout in the paper inserts, the immunoassay device shows great potential in POCT.

The widespread application of electrodialysis is constrained by the high cost of ion exchange membranes, necessitating the development of affordable alternatives. This study focuses on the fabrication and performance evaluation of cation exchange membranes made from polyethersulfone (PES) and sulfonated polyethersulfone (sPES). Membranes were synthesized through phase inversion with varying solvent evaporation times, using N-Methyl-2-Pyrrolidone (NMP) as the solvent. The structural and functional modifications were confirmed using FTIR, XPS, and AFM techniques. Performance tests identified optimal electrodialysis results for PES membranes with a 3 h solvent evaporation time and for sPES membranes with a 1 h evaporation time. Under varying operational conditions, including applied voltage, flow rates, and feed solutions, sPES membranes demonstrated superior performance, underscoring their potential for cost-effective brackish water desalination applications.

This study compares two methods for measuring cell changes: a microfluidic chip single-cell monitoring and a microplate bulk-cell measurement. As intracellular calcium ion concentration ([Ca2+]i) plays a critical role in various cellular functions and biochemical processes, measurements of [Ca2+]i may be used to compare the two methods. The microfluidic approach allows real-time monitoring of individual cells, utilizing the fluorescence emitted from calcium-Fluo 4 chelate, while the microplate method offers bulk analysis of approximately 10,000 cells per well in a 96-well microplate. We have demonstrated that the single-cell method provides insights into [Ca2+]i dynamics with low reagent consumption and rapid analysis, whereas the microplate method enables comprehensive bulk measurements when isolation of single cells is difficult. By integrating both techniques, we aim to complement measurements on both single-cell and population levels, especially when cell availability is an issue. For the cellular process, we specifically investigated the increase in [Ca2+]i following histamine receptor activation, in ACE2-enriched A549 and wild-type A549 cells. In our findings, both approaches yielded consistent calcium-signaling patterns, that wild-type A549 cells exhibited stronger histamine-induced calcium responses than ACE2-enriched cells, and that the two methods complement each other—single-cell assays providing temporal and low-reagent analysis, while bulk assays provide high-throughput, population-level averages.

Pressure-based signal transduction is of promise in developing microfluidic immunoassays such as volumetric bar-chart chips (V-chips), but new working principles are required to further simplify the methods in point-of-care testing (POCT). Herein, we developed immunosyringe sensors and integrated them with bar-chart chips for simple prick-and-read testing of prostate specific antigen (PSA) as a model target. Disposable syringes served as the host for the construction of the sandwich-type immuno-recognition system. Platinum nanoparticles (Pt NPs) as the peroxidase-mimicking detection probe catalyzed the decomposition of H2O2 to produce O2 in the syringe cylinders, enabling the pressure-driven automatic injection of liquids from the syringes. The immuno-recognition event in the syringes was thereby converted into the quantitative autoinjection behavior of the syringes, namely, immunosyringe sensors. By simply pricking the sensors to bar-chart chips, we visually and quantitatively read the immunoassay signals as the bar-chart injection distance of liquids from the syringes in channels of the chips. The immunoassay showed a limit of detection (LOD) of 0.41 ng/mL in PSA detection with satisfactory accuracy in testing clinical serum samples. Owing to the integration with the immunosyringe sensors, this method, in comparison with conventional V-chips, works in a simpler prick-and-read manner without complex chip configurations and specialized chip operations (e.g., on-chip loading of microvolume reagents and sealing treatments). Therefore, the immunoassay shows great potential in POCT applications.

Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentation along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high-throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the PMMA polymer pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich-type immunoassay was performed in the PiPP hybrid device after the optimization of multiple assay conditions. Limits of detection of 0.32 ng mL-1 for carcinoembryonic antigen (CEA) and 0.20 ng mL-1 for prostate-specific antigen (PSA) were obtained, which were about 10-fold better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.

The continuous, noninvasive monitoring of human blood pressure (BP) through the accurate detection of pulse waves has extremely stringent requirements on the sensitivity and stability of flexible strain sensors. In this study, a new ultrasensitive flexible strain sensor based on the interlayer synergistic effect was fabricated through drop-casting and drying silver nanowires and graphene films on polydimethylsiloxane substrates and was further successfully applied for continuous monitoring of BP. This strain sensor exhibited ultrahigh sensitivity with a maximum gauge factor of 34357.2 (∼700% sensitivity enhancement over other major sensors), satisfactory response time (∼85 ms), wide strange range (12%), and excellent stability. An interlayer fracture mechanism was proposed to elucidate the working principle of the strain sensor. The real-time BP values can be obtained by analyzing the relationship between the BP and the pulse transit time. To verify our strain sensor for real-time BP monitoring, our strain sensor was compared with a conventional electrocardiogram-photoplethysmograph method and a commercial cuff-based device and showed similar measurement results to BP values from both methods, with only minor differences of 0.693, 0.073, and 0.566 mmHg in the systolic BP, diastolic BP, and mean arterial pressure, respectively. Furthermore, the reliability of the strain sensors was validated by testing 20 human subjects for more than 50 min. This ultrasensitive strain sensor provides a new pathway for continuous and noninvasive BP monitoring.

The point-of-care testing (POCT) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens and antibodies is of great significance in screening the infection and monitoring the in vivo immunity in controlling the pandemic. However, traditional immunoassays often require advanced laboratory settings with compromised potential in POCT. This article reviews recent advances in microfluidic immunoassays for POCT of SARS-CoV-2 antigens and antibodies. We first briefly introduce different diagnostic methods of coronavirus disease 2019 (COVID-19). After a concise introduction of different microfluidic platforms, we then focus on the latest advances in microfluidic immunoassays for POCT of SARS-CoV-2 antigens and antibodies based on different biosensing principles. In particular, some emerging biosensing principles such as photothermal biosensing are highlighted. At this end, we discuss the current challenges and future perspectives on this topic, which may shed light on new strategies on defense against similar coronavirus and other viruses to avoid pandemics in the future.

Tuberculosis (TB) caused by mycobacterium tuberculosis (M.tb) bacteria is one of the leading causes of death worldwide, with the highest disease burden occurring in developing countries. The existing diagnostic methods either have low sensitivity and specificity or need bulky and expensive instruments, which limit their broad application, especially in resource-limited settings. To address this need, we have developed a paper/polymer hybrid microfluidic platform integrated with loop-mediated isothermal amplification (LAMP) technique for rapid, sensitive, specific, and instrument-free multiplexed-genes detection of M.tb.

A low-cost microfluidic platform integrated with a flexible heater was developed for in situ temperature-dependent spectroscopic measurement at the point of care. After verifying the system by comparing on-chip spectroscopic measurement of methylene blue with the conventional spectroscopy, we demonstrated its applications in temperature-dependent absorption spectroscopy of a model biomolecule, curcumin. The system is portable, battery-powered and requires ultra-low volumes of analytes, which is highly suitable for point-of-care characterization.

Biosensors that sense the concentration of a specified target and produce a specific signal output have become important technology for biological analysis. Recently, intelligent biosensors have received great interest due to their adaptability to meet sophisticated demands. Advances in developing standard modules and carriers in synthetic biology have shed light on intelligent biosensors that can implement advanced analytical processing to better accommodate practical applications. This review focuses on intelligent synthetic biology-enabled biosensors (SBBs). First, we illustrate recent progress in intelligent SBBs with the capability of computation, memory storage, and self-calibration. Then, we discuss emerging applications of SBBs in point-of-care testing (POCT) and wearable monitoring. Finally, future perspectives on intelligent SBBs are proposed.

A disposable paper-based glucose biosensor with direct electron transfer (DET) of glucose oxidase (GOX) was developed through simple covalent immobilization of GOX on a carbon electrode surface using zero-length cross-linkers. This glucose biosensor exhibited a high electron transfer rate (ks, 3.363 s(-1)) as well as good affinity (km, 0.03 mM) for GOX while keeping innate enzymatic activities. Furthermore, the DET-based glucose detection was accomplished by employing both square wave voltammetry and chronoamperometric techniques, and it achieved a glucose detection range from 5.4 mg/dL to 900 mg/dL, which is wider than most commercially available glucometers. This low-cost DET glucose biosensor showed remarkable selectivity, and the use of the negative operating potential avoided interference from other common electroactive compounds. It has great potential to monitor different stages of diabetes from hypoglycemic to hyperglycemic states, especially for self-monitoring of blood glucose.

Microfluidic technology provides a portable, cost-effective, and versatile tool for point-of-care (POC) bioanalysis because of its associated advantages such as fast analysis, low volumes of reagent consumption, and high portability. Along with microfluidics, the application of nanomaterials in biosensing has attracted lots of attention due to their unique physical and chemical properties for enhanced signal modulation such as signal amplification and signal transduction for POC bioanalysis. Hence, an enormous number of microfluidic devices integrated with nano-sensors have been developed for POC bioanalysis targeting low-resource settings. Herein, we review recent advances in POC bioanalysis on nano-sensor-based microfluidic platforms. We first briefly summarized the different types of cost-effective microfluidic platforms, followed by a concise introduction to nanomaterial-based biosensors. Then, we highlighted the application of microfluidic platforms integrated with nano-sensors for POC bioanalysis. Finally, we discussed the current limitations and perspective trends of the nano-sensor-based microfluidic platforms for POC bioanalysis.

Acephate is an organophosphorus pesticide (OP) that is widely used to control insects in agricultural fields such as in vegetables and fruits. Toxic OPs can enter human and animal bodies and eventually lead to chronic or acute poisoning. However, traditional enzyme inhibition and colorimetric methods for OPs detection usually require complicated detection procedures and prolonged time and have low detection sensitivity. High-sensitivity monitoring of trace levels of acephate residues is of great significance to food safety and human health. Here, we developed a simple method for ultrasensitive quantitative detection of acephate based on the carbon quantum dot (CQD)-mediated fluorescence inner filter effect (IFE). In this method, the fluorescence from CQDs at 460 nm is quenched by 2,3-diaminophenazine (DAP) and the resulting fluorescence from DAP at 558 nm is through an IFE mechanism between CQDs and DAP, producing ratiometric responses. The ratiometric signal I558/I460 was found to exhibit a linear relationship with the concentration of acephate. The detection limit of this method was 0.052 ppb, which is far lower than the standards for acephate from China and EU in food safety administration. The ratiometric fluorescence sensor was further validated by testing spiked samples of tap water and pear, indicating its great potential for sensitive detection of trace OPs in complex matrixes of real samples.

Cancer immunotherapy has achieved remarkable success over the past decade by modulating patients' own immune systems and unleashing pre-existing immunity. However, only a minority of cancer patients across different cancer types are able to benefit from immunotherapy treatment; moreover, among those small portions of patients with response, intrinsic and acquired resistance remains a persistent challenge. Because the tumor microenvironment (TME) is well recognized to play a critical role in tumor initiation, progression, metastasis, and the suppression of the immune system and responses to immunotherapy, understanding the interactions between the TME and the immune system is a pivotal step in developing novel and efficient cancer immunotherapies. With unique features such as low reagent consumption, dynamic and precise fluid control, versatile structures and function designs, and 3D cell co-culture, microfluidic tumor organoid-on-a-chip platforms that recapitulate key factors of the TME and the immune contexture have emerged as innovative reliable tools to investigate how tumors regulate their TME to counteract antitumor immunity and the mechanism of tumor resistance to immunotherapy. In this comprehensive review, we focus on recent advances in tumor organoid-on-a-chip platforms for studying the interaction between the TME and the immune system. We first review different factors of the TME that recent microfluidic in vitro systems reproduce to generate advanced tools to imitate the crosstalk between the TME and the immune system. Then, we discuss their applications in the assessment of different immunotherapies' efficacy using tumor organoid-on-a-chip platforms. Finally, we present an overview and the outlook of engineered microfluidic platforms in investigating the interactions between cancer and immune systems, and the adoption of patient-on-a-chip models in clinical applications toward personalized immunotherapy.

Paper-based microfluidic devices have undergone rapid development and offered a promising low-cost platform for disease diagnostics in poor-resource areas. Recently, paper-hybrid microfluidic devices have attracted much attention and been applied in various low-cost point-of-care testings, due to multiple merits derived from both paper and other substrates. This chapter summarizes the recent progress of low-cost paper and paper-hybrid microfluidic devices for rapid diagnostics of human diseases. The commonly used fabrication techniques are first introduced, and the applications of numerous paper and paper-hybrid microfluidic devices are then elaborated with an emphasis on rapid disease diagnostics in terms of the nature of biomolecules in three major categories, namely, protein-, gene-, and cell-based diagnostics. Both advantages and disadvantages of using these devices are discussed, followed by the perspectives for broad applications in low-cost diagnostics.

Flow cytometry is an essential technique in biomedical discovery for cell counting, cell sorting, and biomarker detection. In vivo flow cytometers based on one-photon or two-photon excited fluorescence have been developed for over a decade. One drawback of the laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is limited to the focal spot due to the short Rayleigh range of focused Gaussian beams. Hence, the sampling volume is much smaller than in one-photon flow cytometers, making it challenging to count or detect rare circulating cells in vivo. Non-diffracting light waves like Bessel beams and Airy beams have narrow intensity profiles with an effective spot size as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making them an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Airy beams rather than Gaussian beams is side lobes Airy beams have, which contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using 2D Airy beams to form a light-sheet that intersects the blood vessel a microfluidic channel, which was used to model a blood vessel. The setup can successfully detect and count flowing fluorescent microspheres in a microchannel.

A gold nanoparticle(AuNP)aggregation-induced colorimetric aptasensing method for quantitative detec-tion of sulfadimethoxine(SDM)with a smartphone was developed.AuNPs were complexed with aptamers which protected AuNPs from aggregating in high-concentration salt solutions.In the presence of SDM,SDM bound with the aptamer on the surface of AuNPs with higher affinity,which competitively des-orbed the aptamer from the AuNP surface and resulted in AuNPs aggregation,accompanied with a color change from red to purple-blue.The R,G and B values of images taken by a smartphone camera were analyzed with an app on the smartphone,and were utilized for quantitative analysis of SDM.Under the optimized conditions,the colorimetric aptasensing method using a smartphone showed high sensitivity for SDM,with the limit of detection of 0.023 ppm,lower than the allowed maximum SDM residue limit.This study provides a simple,fast,and easy to read method for on-site quantitative biochemical and cel-lular analysis.