Recombinase polymerase amplification (RPA)-CRISPR/Cas12a assays have demonstrated remarkable potential for point-of-care detection of pathogens in resource-limited settings. Nevertheless, these assays fall short in delivering direct quantitative results due to the incompatibility between the RPA and CRISPR/Cas12a systems. To overcome this limitation, we developed a droplet pairing-merging enabled digital RPA-CRISPR/Cas12a (DIMERIC) assay in this study. By leveraging a microfluidic chip with a calabash-shaped microwell array, large-volume RPA droplets and small-volume CRISPR/Cas12a droplets were sequentially and size-selectively trapped, generating one-to-one droplet pairs. This spatial separation of the droplets eliminates the inhibitory effects of the CRISPR/Cas12a chemistry on RPA. Upon the completion of RPA, the CRISPR/Cas12a system can be activated by merging the paired droplets. This temporal separation of the RPA and CRISPR/Cas reactions allows for the accumulation of sufficient amplicons to efficiently unleash the collateral cleavage activity. The DIMERIC assay offers rapid quantification of nucleic acids, with the entire procedure being accomplished within 20 min. This assay was employed for the quantitative detection of Hepatitis B virus DNA from batched clinical serum samples, demonstrating a good correlation with qPCR (R2 = 0.92033) and ddPCR (R2 = 0.97337) outcomes. Consequently, the developed DIMERIC assay provides a valuable tool for rapid and precise quantification of pathogenic nucleic acids.

The diamond-shaped platinum (II) complexes have demonstrated remarkable electrochemiluminescence (ECL) properties. However, their broader application in ECL sensing has been limited by their relatively poor solubility in aqueous solutions and an insufficient understanding of their ECL emission mechanism. In this study, we synthesized an amphiphilic anionic platinum(II) bzimpy complex PS-BZIMPY-Pt, where the introduction of two sulfonate groups significantly improved its aqueous solubility. Notably, this complex exhibits typical aggregation-induced emission (AIE) and aggregation-induced electrochemiluminescence (AIECL) properties. Its aggregate reveals intense anodic and cathodic ECL emissions, with ECL efficiencies of 921.58 % and 6939.31 % respectively, using Ru(bpy)32+ as a reference standard. Due to the suppressed formation of sulfate radicals (SO4 center dot-) by glutathione (GSH) during the cathodic ECL process, thereby quenching the ECL emission of PS-BZIMPY-Pt, an ECL sensor for detecting GSH was developed with a broad linear detection range of 0.1-200 mu M, a low limit of detection of 0.016 mu M. Additionally, the further application of this sensor for screening of glutathione reductase (GR) activity was validated. This study explored the ECL properties of the aqueous soluble platinum(II) complex, particularly, its ECL emission mechanism was delved, which may extensively broaden the potential application of platinum complexes in ECL sensing.

A nano-foam gold/BDD aptasensor achieves femtomolar 17β-estradiol detection in water via nanostructural and molecular synergy, enabling selective environmental monitoring.

The etiological diagnosis of severe infectious diseases is complicated by variability in the timing of sample submissions, heterogeneity in detection targets, and stringent requirements for rapid turnaround times, thereby necessitating the development of on-demand detection methodologies. To address these challenges, we introduce an automated digital isothermal nucleic acid amplification (dINA) assay facilitated by a digital-to-droplet (D2D) microfluidic platform. This D2D system permits the continuous introduction and extraction of water-in-oil droplets, enabling the digital microfluidics (DMF) module to alternately interface with multiple droplet microfluidic units. Within this framework, samples or reagents encapsulated in droplets are sequentially delivered to the DMF module, where nucleic acid extraction and subsequent mixing with loop-mediated isothermal amplification (LAMP) reagents are accomplished through a series of precise droplet manipulations. The resultant microliter-scale LAMP reaction droplets are then dispensed into the sample inlets of individual droplet microfluidic modules, where they are partitioned into picoliter-scale droplets for dINA analysis. The principal advantages of this system include (1) the ability to process samples immediately upon receipt, (2) substantial scalability in throughput, and (3) absolute quantification independent of calibration standards. As a proof of concept, this platform was employed to detect viruses responsible for post-transplantation infections, demonstrating a markedly reduced turnaround time for specimens submitted at variable intervals. These findings underscore the potential of this system as an efficient and adaptable diagnostic tool for the on-demand detection of pathogens implicated in severe infectious diseases.

Luminol is a well-known electrochemiluminescence (ECL) fluorophore that is applied in various sensing fields as an ECL reporter. Regulating the signal off/on transition of an ECL fluorophore offers great opportunities for sensors' design; however, such attempts on luminol are extremely scarce as it was regarded to lack promising modification sites. In this study, we developed four luminol derivatives with modification at the amine site and the enol site and systematically explored possible caging strategies to regulate ECL emission. The electrochemical and corresponding ECL properties reveal that the analogue with etherification on the enol group significantly inhibits the electrochemical oxidation behavior of luminol and almost entirely silences its ECL emission. Inspired by this observation, a new ECL probe luminol-O-β-d-glucose (Lum-Glc) was prepared by integrating glucose moieties and luminol through an ether bond. The ether linkage quenched the ECL emission of Lum-Glc, which could be cleaved by β-glucosidase (β-Glu), enabling the "turn-on" ECL determination of β-Glu. A broad linear detection range of 0.4-400 U/L with a low limit of detection of 0.1 U/L was achieved. This study provides new insight into mediating the ECL performance of luminol for the design of luminol-based ECL biosensors.

The rapid, precise, and automated diagnosis of infectious diseases is crucial for effective disease management and control. Herein, the integrated portable and automatic digital detection system (IPADS), a novel diagnostic platform for nucleic acid detection is introduced. The device employs the hybrid magnetic system (HMS), which uses an electromagnet and a movable permanent magnet to modulate the magnetic field and control bead movement, increasing nucleic acid extraction efficiency to over 80%, while simplifying the traditional labor-intensive process and enabling quick, low-risk sample processing. Additionally, a disposable cartridge is designed for integrated HMS based preprocessing, with detection performed using digital RPA-Cas12a, enabling rapid, enclosed, and automation-friendly detection across a dynamic range spanning five orders of magnitude, with a sensitivity as low as 100 copies mL-1 in serum samples. An automated platform further optimizes workflow. As a proof of concept, IPADS is applied to detect hepatitis B virus (HBV) DNA in 20 clinical serum samples, demonstrating high concordance with gold-standard quantitative PCR (qPCR) methods. These results validate the potential of IPADS as a reliable point-of-care testing solution.

Optical endoscopy, as one of the common clinical diagnostic modalities, provides irreplaceable advantages in the diagnosis and treatment of internal organs. However, the approach is limited to the characterization of superficial tissues due to the strong optical scattering properties of tissue. In this work, a microwave-induced thermoacoustic (TA) endoscope (MTAE) was developed and evaluated. The MTAE system integrated a homemade monopole sleeve antenna (diameter = 7 mm) for providing homogenized pulsed microwave irradiation to induce a TA signal in the colorectal cavity and a side-viewing focus ultrasonic transducer (diameter = 3 mm) for detecting the TA signal in the ultrasonic spectrum to construct the image. Our MTAE, system combined microwave excitation and acoustic detection; produced images with dielectric contrast and high spatial resolution at several centimeters deep in soft tissues, overcome the current limitations of the imaging depth of optical endoscopy and mechanical wave-based imaging contrast of ultrasound endoscopy, and had the ability to extract complete features for deep location tumors that could be infiltrating and invading adjacent structures. The practical feasibility of the MTAE system was evaluated i n vivo with rabbits having colorectal tumors. The results demonstrated that colorectal tumor progression could be visualized from the changes in electromagnetic parameters of the tissue via MTAE, showing its potential clinical application.

Programmable droplet microfluidics (PDM) refers to a microfluidic device that integrates the functionality of a series of droplets with distinct spatial locations in a designated temporal order. PDM streamlines the intricate workflow of complex bioassays by enabling programmable and macroscopic droplet displacements, in which the droplets serve as reservoirs for reagents, microvalves for liquid insulation, and in some cases micropumps for mass transportation. As these droplets are intangible structures, the need for expensive microfabrication procedures is eliminated. Furthermore, the parallelization of the droplet series provides flexibility in controlling the throughput of the microfluidic analysis system. This paper provides a comprehensive review of PDMs enabled by various microfluidic mechanisms, including magnetism actuation, relative liquid displacement, capillary suction and sequential microdisplacement. Additionally, important applications of PDM systems for nucleic acid detection, immunoassay, drug testing, and sample recovery are also introduced. In conclusion, PDM demonstrates its potential as a highly advantageous tool for executing intricate multistep bioassays on a microfluidic platform. These technologies have exhibited superiority over their traditional counterparts in terms of size reduction, automation, and low sample/reagent consumption.

Early and rapid diagnostic of acute myocardial infarction (AMI) during its developing stage is crucial due to its high fatality rate. Heart-type fatty acid binding protein (h-FABP) is an ideal biomarker for the quantitative diagnosis of AMI, surpassing traditional markers such as myoglobin, creatine phosphokinase-MB, and troponin in terms of sensitivity, specificity, and prognostic value. To obtain diagnostic and prognostic information, a precise and fully quantitative measurement of h-FABP is essential, typically achieved through an immunosorbent assay like the enzyme-linked immunosorbent assay. Nevertheless, this method has several limitations, including extended detection time, complex assay procedures, the necessity for skilled technicians, and challenges in implementing automated detection. This research introduces a novel biosensor, utilizing aggregation-induced emission nanoparticles (AIENPs) and integrated with a digital microfluidic (DMF) workstation, designed for the sensitive, rapid, and automated detection of h-FABP in low-volume serum samples. AIENPs and magnetic beads in nanoscale were served as the capture particles and the fluorescent probe, which were linked covalently to anti-h-FABP antibodies respectively. The approach was based on a sandwich immunoassay and performed on a fully automated DMF workstation with assay time by 15 min. We demonstrated the determination of h-FABP in serum samples with detection limit of 0.14 ng/mL using this biosensor under optimal condition. Furthermore, excellent correlations (R2 = 0.9536, n = 50) were obtained between utilizing this biosensor and commercialized ELISA kits in clinical serum detecting. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for AMI patients.

Disulfiram (DSF) and copper (Cu2+) in combination exhibit powerful anti-cancer effect on a variety of cancer cell lines. Here, we found that DSF/Cu2+ facilitated the accumulation of intracellular reactive oxygen species (ROS), and induced ROS-dependent apoptosis accompanied by chromatin condensation and phosphatidylserine externalization in MCF-7 cells. DSF/Cu2+ caused caspase-independent apoptosis by promoting the AIF translocation from mitochondria to nucleus. Most importantly, the cytotoxicity of DSF/Cu2+ was markedly inhibited by knocking out AIF, suggesting the indispensability of AIF in DSF/Cu2+-induced apoptosis. The pro-apoptotic protein BAK instead of BAX was upregulated and activated upon DSF/Cu2+ treatment, and the BAK knockout cells exhibited high resistance to DSF/Cu2+, indicating the importance of BAK in DSF/Cu2+-induced apoptosis. Additionally, both co-immunoprecipitation and live-cell quantitative fluorescence resonance energy transfer (FRET) analysis revealed that DSF/Cu2+ unlocked the binding of MCL-1 to BAK, which resulted in subsequent BAK homo-oligomerization. Overall, our data demonstrate for the first time that DSF/Cu2+ unlocks the binding of MCL-1 to BAK, thus leading BAK oligomerization and subsequent AIF nucleus translocation to mediate caspase-independent apoptosis in MCF-7 cells.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

BACKGROUND:Microwave-induced thermoacoustic (TA) imaging (MTAI) is a promising alternative to biomedical imaging due to its high resolution, deep imaging depth, and minimal biohazard. To provide images of different anatomical regions and apply them to different clinical scenarios, the development of miniaturized portable TA probes is imperative.PURPOSE:This study is aimed to propose a highly efficient handheld non-reflective microwave-acoustic coaxial TA probe to advance the translation of MTAI for the clinical use.METHODS:The TA probe integrates a hollowed microwave antenna with a forward radiating uniform microwave field and a linear ultrasonic transducer array which is placed in the hole in the middle of the antenna that has almost no effect on the microwave distribution. The integrated probe was evaluated for properties, including the excitation efficiency of the microwave and the reception efficiency of the acoustic signal. Finally, an isolated EMT6 cell tumor was embedded in a sheep mammary gland to simulate the natural breast tumor environment, and the tumor was detected with the proposed MTAI probe to evaluate its practical feasibility.RESULTS:Compared with the previous TA imaging probe, it has improved the detection efficiency of TA signals by up to 41%, contributing to an improved signal-to-noise ratio (SNR) of the image. The proposed TA probe successfully detected tumors embedded in the breast with a contrast ratio 3.27 times higher than the surrounding tissue in phantom experiments.CONCLUSION:The proposed TA probe with the features of microwave illumination and ultrasonic detection of coaxial, avoiding TA signal attenuation due to reflection, enables high-efficient TA signal excitation and detection. The proposed TA probe is essential for improving the excitation and detection efficiency of TA signals, and increasing the flexibility of the probe, providing a bright future for the clinical application of MTAI technology.

Carbapenem-resistant organisms (CROs) are characterized by high drug resistance, rapid transmission, and high lethality. Therefore, rapid detection for CROs is essential for appropriate applying antibiotics and implementing quarantine. Droplet digital chromogenic assays (DDCA) have been accepted as an effective means for rapid microbial detection as the small droplet volumes facilitate a significant enhancement in the local concentration of chromogenic factors and, therefore, reduce the required time of the test. Nevertheless, as their dependence on the time-consuming isolation culture, the DDCA is still associated with a long turnaround time. To overcome this limitation, we develop here a microfluidic chip-based CRO phenotypic identification method that integrates cascade filtration (CF) with DDCA (CF-DDCA). After a body fluid sample is introduced to the microfluidic chip, particles with sizes >5 mu m are removed out by the primary filter, and Gram (+) cocci with sizes <1 mu m removed out by the secondary filter so that only Gram (-) bacilli with sizes between 1.5 and 5 mu m are selectively retained. The purified Gram (-) bacilli, along with chromogenic reagents and carbapenem antibiotics, are then subjected to the DDCA. We demonstrate that the CF can remove 99.9% of the interfering microorganisms and thus eliminates the isolating culture. Benefited from the isolating culture-free DDCA, phenotypic identification of CROs can be achieved within 3.5 h. Clinical urine sample testing shows that the sensitivity and specificity of the CF-DDCA for CRO identification are all 100%, and the total coincidence rate between CF-DDCA and the con-ventional assay is also 100%.

Microwave thermoacoustic imaging (MTAI) is an exciting imaging technique rooted from the underlying principle of exploiting the distinct electrical properties of biological tissues. By using short-pulsed microwaves as a stimulation source and their interaction with the human body, MTAI has paved the way for revolutionary advancements in medical imaging. When microwaves are absorbed by polar molecules and ions within the tissues, an ingenious thermoelastic effect gives rise to ultrasound waves. These ultrasound waves, brimming with invaluable pathological and physiological insights, propagate outward, carrying the essence of the composition and functionality of biological tissue. Through a meticulous collection of ultrasound signals from all directions surrounding the tissue, it becomes possible to reconstruct intricate internal structures and visualize the tissue's functional dynamics. The MTAI excels in non-invasiveness, capable of delving several centimeters beneath the surface with a microscopic resolution on the order of micrometers. The magic lies in converting microwave energy into ultrasound waves, entering into the hidden depths of tissues without causing harm. This groundbreaking imaging modality unlocks a realm of possibilities for acquiring profound insights into the intricate structures and functionality of deep-seated tissues. Furthermore, the inherent polarization characteristics of microwaves empower MTAI to capture additional dimensions of information, unraveling the intricate polarization properties and illuminating a richer understanding of the tissue's complexity. The great potential of MTAI extends far and wide within the medicine field. It has made remarkable achievements in non-invasive imaging of brain structures, screening breast tumors, visualizing human arthritis, and detecting liver fat content. These accomplishments have laid a solid foundation, firmly establishing MTAI as a trailblazing medical imaging technique. The present study offers a comprehensive and in-depth exploration of the physical principles underpinning MTAI, the sophisticated system devices involved, and the recent groundbreaking research breakthroughs. Moreover, it delves into the exciting prospects and challenges that lie ahead in the future development of MTAI. As the technology continues to progress by leaps and bounds, MTAI is ready to break down barriers, and usher in a new era of unmatched imaging quality and performance. This, in turn, will open the floodgates for transformative innovations and applications in medical diagnosis and treatment. The anticipation is palpable as MTAI strives to make substantial contributions to the ever-developing medical imaging field, bestowing upon humanity more accurate, reliable, and life-enhancing diagnostic capabilities.

Despite the advantages of digital nucleic acid analysis (DNAA) in terms of sensitivity, precision, and resolution, current DNAA methods commonly suffer a limitation in multiplexing capacity. To address this issue, a droplet encoding-pairing enabled DNAA multiplexing strategy is developed, wherein unique tricolor combinations are deployed to index individual primer droplets. The template droplets and primer droplets are sequentially introduced into a microfluidic chip with a calabash-shaped microwell array and are pairwise trapped and merged in the microwells. Pre-merging and post-amplification image analysis with a machine learning algorithm is used to identify, enumerate, and address the droplets. By incorporating the amplification signals with droplet encoding information, simultaneous quantitative detection of multiple targets is achieved. This strategy allows for the establishment of flexible multiplexed DNAA by simply adjusting the primer droplet library. Its flexibility is demonstrated by establishing two multiplexed (8-plex) droplet digital loop-mediated isothermal amplification (mddLAMP) assays for individually detecting lower respiratory tract infection and urinary tract infection causative pathogens. Clinical sample analysis shows that the microbial detection outcomes of the mddLAMP assays are consistent with those of the conventional assay. This DNAA multiplexing strategy can achieve flexible high-order multiplexing on demand, making it a desirable tool for high-content pathogen detection.

As a reliable signaling biomolecule for oxidative stress, the accurate hypochlorous acid (HClO) detection during agent-stimulated oxidative stress plays a vital role in pathological and physiological mechanism exploration for disease theranostics. Therefore, the development of robust analytical tools for HClO is highly significant. Herein we presented an ESIPT fluorescent probe (MEBTA-Cl) for HClO detection. MEBTA-Cl exhibited ultrarapid-response (around 6 s), high sensitivity (42-folds) and low detection limit (7.8 nM) toward HClO titration in solution. MEBTA-Cl was capable to detect both exo-/endogenous HClO in living cells. Moreover, MEBTA-Cl was successfully employed to monitor HClO level in acetaminophen (APAP), disulfiram (DSF) and doxorubicin (DOX)-stimulated oxidative stress. Importantly, using this useful probe, it was available to detect HClO in li-popolysaccharides (LPS) and APAP treated zebrafish.

As a new type of carbon material, graphdiyne (GDY) has wide application prospects in the fields of photocatalysts and photoelectrochemical detection due to its suitable energy bandwidth and high electronic conductivity. However, the highly conjugated and stable macrocyclic structure of GDY makes it difficult to be isomerized and modified on other materials. In this work, click chemistry was used to successfully carboxylate GDY so that it can be stably combined with other optoelectronic materials and some small biological molecules through chemical bonds. A composite optoelectronic material of carboxylated GDY and TiO2 was synthesized and successfully applied in the field of photoelectrochemical detection, a stable and sensitive dopamine sensor was obtained. Based on multiple signal amplification strategy, a wide linear detection range from 0.0005 mM to 1.05 mM with a low detection limit (1.36 x 10-4 mM) was obtained.(c) 2022 Elsevier Ltd. All rights reserved.

Hydrogen peroxide (H2O2) is closely related to a variety of human diseases. It is a challenge to dynamically monitor biological H2O2 activity. Hence, we design and prepare a NIR-emitting ratiometric fluorescent probe (HBQ-B) for detecting H2O2 according to the excited-state intramolecular proton transfer (ESIPT) mechanism using the benzyl bomnic pinacol ester group as a recognition unit. HBQ-B displayed specificity toward H2O2 over other bioactive analytes. Meanwhile, HBQ-B exhibited a good linear relationship between the fluorescence ratio (I-656/I-530) changes and the concentrations of H2O2 (0-10 mu M). HBQ-B also showed a large Stokes shift (234 nm) and a marked detection limit (40.2 nM). HBQ-B was utilized to image the intracellular H2O2 in living MCF-7 cells, HeLa cells and macrophages (RAW 264.7 cells), respectively. In addition, HBQ-B was used to monitor the dynamics of H2O2 level changes during zebrafish development.

Digital nucleic acid analysis is a favorable tool for rapid molecular detection of pathogens due to its absolute quantification capability without the necessity of standard curves. However,current digital nucleic acid analysis platforms commonly perform the workflow with discrete facilities,which complicates the operation process and lengthens the turnaround time,therefore restricting its adoption in resource-limited settings. To address this issue,an integrated microfluidic droplet digital isothermal amplification system was developed in this work. This system integrated sequential droplets-based nucleic acid extraction unit,syringe-vacuum actuated droplet generation unit,and droplet digital loop-mediated isothermal amplification (LAMP)unit, achieving digital analysis of bacterial nucleic acid within 1.5 h in an integrated manner. The nucleic acid extraction unit could complete E. coli genomic DNA extraction with the efficiency of 93.68% & PLUSMN;32.38%,the droplet generation unit could produce 20000 droplets in 4 min with the relative standard deviation (RSD)less than 10%. The LAMP reaction could be performed with a linear dynamic range of 4 orders of magnitude (2.36 x 104 - 1.71 x 1 07 CFU/ mL). Compared to traditional culture method,the results obtained from analysis of E. coli in UTI clinical samples (n =13)showed that both the detection sensitivity and specificity of this digital nucleic acid analysis method were 100% (Kappa= 1,p < 0.01). With the advantages of accurate quantification and ease of operation,this integrated microfluidic droplet digital isothermal amplification system was expected to be a favorable tool for rapid point-of-care detection of pathogens in resource-limited settings.

Early screening of cancer can effectively prolong survival time and reduce cancer mortality. However, the existing health-monitoring devices can only be carried out in professional laboratories, so large-scale early cancer screening in resource-limited settings is hardly achieved. To embrace the challenge, we developed a novel chemiluminescence immunoassay (CLIA) analyzer that does not require a professional operation. Then, it was applied to detect carbohydrate antigen 50 (CA50), a non–organ-specific tumor marker for screening various cancers. As a result, the analyzer exhibited excellent performance that the total assay time was only 15 min, and the detection limit reached 0.057 U ml−1. A coefficient of variance (CV) less than 15% was well-controlled for both intra- and inter-assay precision, and the linear range was 0–500 U ml−1. More importantly, this analyzer can continuously detect 60 samples per hour without any professional paramedic. Finally, this analyzer has been applied to evaluate clinical samples and the detected results showed a good correlation with the clinical test results (correlation coefficient, 0.9958). These characteristics exactly meet large-scale and high-throughput early screening of cancer. Thus, this miniaturized analyzer for CA50 detection is promising to achieve early large-scale screening of cancer in the resource-limited grassroots community.