CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7.

Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R2 = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.

Development of a hybridization chain reaction-powered lab-on-fiber device for ultrafast point-of-care testing of circulating tuor DNA in whole blood.

Circulating tumor DNA (ctDNA) demonstrates great promise in the guidance of prognostication, diagnosis, and surveillance of cancers, which highlights the need for rapid and sensitive point-of-care testing (POCT) technologies. Hybridization chain reaction (HCR)-based optical biosensors provide excellent solutions due to their prominent features. However, the requirement of a sophisticated and expensive optical readout device, relatively long detection time, and heating hold back their scalability and clinical applications. Here, an innovative HCR-powered lab-on-fiber device (HCR-LOFD) was developed for rapid on-site detection of ctDNA with high sensitivity, specificity, and reproducibility. A LOFD with a compact all-fiber optical structure was constructed for the fluorescence detection of the HCR system. Combining HCR, fluorescence energy resonant transfer, and the evanescent wave fluorescence principle, HCR-LOFD achieved the quantitative detection of KRAS G12D, the 12th amino acid from glycine (Gly) mutated aspartate (Asp) and the most common mutation of KARS, in 5 min at room temperature based on end-point detection mode or real-time fluorescence detection mode. This new assay platform was also successfully applied for the direct detection of KRAS G12D in whole blood with simple dilution. The application of HCR-LOFD not only greatly simplifies the complexity of optical readout devices and improves their scalability but also potentially serves as a sample-to-answer solution for the detection of biomarkers in limited medical resource regions.

CRISPR/Cas12a-powered evanescent wave fluorescence nanobiosensing platform for nucleic acid amplification-free detection of Staphylococcus aureus with multiple signal enhancements.

Although CRISPR-based biosensors for pathogenic detection are highly specific, they not sensitive enough and nucleic acid amplification is generally required to improve their sensitivity. However, this allows only binary operations and significantly limits practical applications. Here, a CRISPR/Cas12a-powered Evanescent wAve fluorescence nanobiosensing plaTform (CREAT) was developed for ultrasensitive nucleic acid amplification-free quantitative detection of pathogens with multiple signal enhancements. In addition to collateral cleavage amplification of the CRISPR/Cas12a system, we constructed nanophotonic structure-based evanescent wave fluorescence enhancement, Mg2+ or DNA-mediated fluorescence enhancement, and air-displacement fluorescence enhancement strategies for ultrasensitive detection of Staphylococcus aureus (S. aureus). Especially, the fluorescence signal detected by CREAT can be significantly enhanced by adding a simple air displacement step, thus improving detection sensitivity. This nanobiosensor detected real samples containing S. aureus, with a detection limit of 592 CFU/mL and 13.2 CFU/mL in 45 min and 90 min, respectively, which are comparable to those of RT-qPCR. This paves a new way for simple, rapid, sensitive, robust, and flexible on-site detection of S. aureus as well as other pathogens.

Universal and rapid detection of atrazine and bisphenol A using a reusable optical fiber chemiluminescent biosensor.

Timely and accurately detection of small molecule pollutants is quite necessary to control environmental pollution and reduce harmfulness. Herein, a reusable optical fiber chemiluminescent biosensor (ROFC) was proposed for universal and rapid detection of two representative pollutants, pesticide atrazine (ATZ) and endocrine disruptor bisphenol A (BPA). The optical fiber modified with hapten-protein conjugates was regarded as both bio-probe and chemiluminescence signal transmission element, which effectively improved the light transmission efficiency and signal-to-noise ratio of the system. High-sensitive chemiluminescence signal detection is realized with a miniaturized ultrasensitive photodiode detector. Good regeneration performance of bio-probe can reduce detection cost and ensure detection reproducibility. Based on indirect competitive immunoassay principle, the chemiluminescence signal decreased with increasing pollutant concentration resulting from the less amount of antibody combined on the bio-probe surface. Under optimal conditions, the whole assay was achieved within 25 min with linear range of 1-100 µg/L and detection limits (LOD) for atrazine and BPA are 0.029 µg/L and 0.025 µg/L, respectively. The immunosensing optical fiber probe can be reused for 150 times at least without losing obvious bioactivity. The method was successfully applied to the detection of ATZ and BPA in three environmental samples, where recoveries between 93.4% and 116.6% were achieved. The ROFC biosensor provides a feasible platform for rapid detection of multiple small molecule pollutants in the environment.

Development of chemiluminescent lab-on-fiber immunosensors for rapid point-of-care testing of anti-SARS-CoV-2 antibodies and evaluation of longitudinal immune response kinetics following three-dose inactivation virus vaccination.

Developing reliable, rapid, and quantitative point-of-care testing (POCT) technology of SARS-CoV-2-specific antibodies and understanding longitudinal vaccination response kinetics are highly required to restrain the ongoing coronavirus disease 2019 (COVID-19) pandemic. We demonstrate a novel portable, sensitive, and rapid chemiluminescent lab-on-fiber detection platform for detection of anti-SARS-CoV-2 antibodies: the chemiluminescent lab-on-fiber immunosensor (c-LOFI). Using SARS-CoV-2 Spike S1 RBD protein functionalized fiber bio-probe, the c-LOFI can detect anti-SARS-CoV-2 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies with high sensitivity based on their respective horseradish peroxidase-labeled secondary antibodies. The limits of detection of anti-SARS-CoV-2 IgG and IgM antibodies were 0.6 and 0.3 ng/ml, respectively. The c-LOFI was successfully applied for direct detection of anti-SARS-CoV-2 antibodies in whole blood samples with simple dilution, which can serve as a finger prick test to rapidly detect antibodies. Furthermore, the longitudinal immune response (>12 months) kinetics following three-dose inactivated virus vaccines was evaluated based on anti-SARS-CoV-2 IgG detection results, which can provide important significance for understanding the immune mechanism against COVID-19 and identify individuals who may benefit from the vaccination and booster vaccination. The c-LOFI has great potential to become a sensitive, low-cost, rapid, high-frequency POCT tool for the detection of both SARS-CoV-2-specific antibodies and other biomarkers.

Development of portable whole-cell biosensing platform with lyophilized bacteria and its application for rapid on-site detection of heavy metal toxicity without pre-resuscitation.

The high toxicity of heavy metals necessitates monitoring technology that allows rapid adaptation and deployment. Microbial whole-cell biosensors have become a priority because of their excellent performance. However, traditional methods have several limitations, including long assay time, poor portability, and the lack of ready-to-use on-site devices. In this study, a novel portable whole-cell biosensing platform was developed by integrating a simple handheld fiber-optic dissolve oxygen sensor and bacterial culture or lyophilized bacteria. Based on the oxygen consumption inhibition mechanism, this platform achieved rapid acute toxicity measurement of heavy metal ions through inhibit Escherichia coli (E.coli) respiration. Under the optimal conditions, the limit of detection and IC50 of Hg2+ using E. coli culture were 5.62 µM and 11.64 µM, respectively. Interestingly, the lyophilized E. coli could be directly applied for Hg2+ toxicity detection without pre-resuscitation, where an IC50 of 31.28 µM was obtained, and the whole detection process was only 18 min. The lyophilized E. coli could be stored long-term at -80 °C without significant loss of activity and detection performance. The portable whole-cell biosensing platform demonstrated a high potential for rapid on-demand toxicity detection in real water samples. The developed strategy is not only fast, portable, and easily storable, but also highly suited for on-site ready-to-use, and high-frequency toxicity detection of heavy metal ions in the field.

On-site rapid and simultaneous detection of acetamiprid and fipronil using a dual-fluorescence lab-on-fiber biosensor.

A dual-fluorescence lab-on-fiber biosensor was developed for the rapid and simultaneous on-site determination of acetamiprid and fipronil, based on time-resolved effect and indirect competitive immunoassay principle. The optical fiber modified with two hapten-protein conjugates serves as a bifunctional bio-probe. The dual-color fluorescent reporters were prepared via labeling acetamiprid and fipronil antibodies with Cy5.5 and Alexa Fluor 555, which were excited at 635-nm and 520-nm laser wavelengths, respectively. In the presence of targets, the binding sites of corresponding antibodies were occupied and less antibodies were connected to the probe surface, resulting in the reduction of fluorescence signal. The concentration of acetamiprid and fipronil was determined by measuring the fluorescence signals at 568 nm and 702 nm (emission wavelengths), respectively. Under optimal conditions, the linear response range was 14.2-225.4 ng/L for acetamiprid and 25.1-162.8 ng/L for fipronil, and the limit of detection was 6.51 ng/L and 17.8 ng/L for acetamiprid and fipronil, respectively. The method was successfully applied to the simultaneous detection of acetamiprid and fipronil in three environmental samples, and the recoveries were between 90 and 128%. The dual-fluorescence lab-on-fiber biosensor provides a feasible platform for simultaneous and rapid detection of multiple pesticide residues. A dual-fluorescence lab-on-fiber biosensor was developed for the rapid and simultaneous on-site determination of acetamiprid and fipronil. A bifunctional bio-probe was prepared from the optical fiber modified with two hapten-protein conjugates. Acetamiprid and fipronil antibodies were labeled with different fluorophores and used as dual-color fluorescent reporters.

Rapid, Sensitive On-Site Detection of Deoxynivalenol in Cereals Using Portable and Reusable Evanescent Wave Optofluidic Immunosensor.

This paper develops an improved portable and reusable evanescent wave optofluidic immunosensor (OIP-v2) for rapid and sensitive on-site determination of deoxynivalenol (DON), one of the most frequently detected mycotoxins mainly produced by Fusarium species. Using the bifunctional reagent N,N'-Disuccinimidyl carbonate, deoxynivalenol-bovine-serum-albumin (DON-BSA) were covalently modified onto a bio-probe surface as biorecognition elements, whose robustness allowed it to perform multiple detections without significant activity loss. An indirect competitive immunoassay strategy was applied for DON detection. Under optimal conditions, the limit of detection of 0.11 µg/L and the linear dynamic detection range of 0.43 to 36.61 µg/L was obtained when the concentration of the Cy5.5-anti-DON antibody was 0.25 µg/mL. The OIP-v2 was also applied to detect DON in various cereals, and the recoveries ranged from 81% to 127%. The correlation between OIP-v2 and enzyme-linked immunosorbent assay (ELISA) through the simultaneous detection of maize-positive samples was in good agreement (R2 = 0.9891).

Rapid and quantitative detection of SARS-CoV-2 IgG antibody in serum using optofluidic point-of-care testing fluorescence biosensor.

The COVID-19 pandemic brings unprecedented crisis for public health and economics in the world. Detecting specific antibodies to SARS-CoV-2 is a powerful supplement for the diagnosis of COVID-19 and is important for epidemiological studies and vaccine validations. Herein, a rapid and quantitative detection method of anti-SARS-CoV-2 IgG antibody was built based on the optofluidic point-of-care testing fluorescence biosensor. Without complicated steps needed, the portable system is suitable for on-site sensitive determination of anti-SARS-CoV-2 IgG antibody in serum. Under the optimal conditions, the whole detection procedure is about 25 min with a detection limit of 12.5 ng/mL that can well meet the diagnostic requirements. The method was not obviously affected by IgM and serum matrix and demonstrated to have good stability and reliability in real sample analysis. Compared to ELISA test results, the proposed method exhibits several advantages including wider measurement range and easier operation. The method provides a universal platform for rapid and quantitative analysis of other related biomarkers, which is of significance for the prevention and control of COVID-19 pandemic.

Development of a novel label-free all-fiber optofluidic biosensor based on Fresnel reflection and its applications.

A novel, compact, cost-effective, and robust label-free all-fiber optofluidic biosensor (LF-AOB) based on Fresnel reflection mechanism was built through integrating single-multi mode fiber coupler and highly sensitive micro-photodetector. The Fresnel reflection light intensity detected by the LF-AOB greatly depended on the RI change on the end-surface of the fiber probe according to experimental and simulation results. The capability of the LF-AOB for real-time in situ detection in optofluidic system were verified by measuring salt and protein solution, and the lowest limit of detection was 1.0 × 10-6 RIU. Our proposed theory can effectively eliminate the influence of light intensity fluctuation, and one-point calibration method of sensor performance is conducive for rapid and convenient detection of targets. Label-free sensitive detection of SARS-Cov-2 Spike protein receptor-binding domain (S-RBD) and the binding kinetics assay between S-RBD and anti-S-RBD antibody were achieved using the LF-AOB. These contributed to the elegant design of all-fiber optical system with high efficiency, high resolution and sensitivity of micro-photodetector, and enhanced interaction between the light and the samples at the liquid-sensor interface because of the large surface area of the multi-mode fiber probe. The LF-AOB can be extended as a universal sensing platform to measure other factors associated with refractive index because its high sensitivity, low sample consumption (∼160 nL), and capability of real-time in situ detection.

Portable and automated fluorescence microarray biosensing platform for on-site parallel detection and early-warning of multiple pollutants.

The portable and automated fluorescence microarray biosensing platform (FMB) that employed a compact hybrid optical structure, microfluidics, and microarray biosensors was constructed for on-site parallel detection of multiple analytes. In the FMB, a hybrid optical structure that composed of a 1 × 4 single mode fiber optic coupler, four fiber optic switches, a single-multi mode fiber optic bundle coupler was at the first time developed for the transmission of the excitation light and the collection and transmission of multi-channel fluorescence signals. Through the control of fiber optic switches, the parallel fluorescence assay of four channels could be achieved using only one excitation light and one photodiode detector on the basis of the time-resolved effect. This optical design not only greatly increased the efficiency of light transmission and fluorescence collection and detection sensitivity of the FMB, but also allows the miniaturization and portability of the whole system because of few optical separation elements used and no requirement of rigorous optical alignment. Taking Microcystin-LR (MC-LR), 2,4-D, atrazine (ATZ), and bisphenol A (BPA) for example, the application potential of the FMB to rapidly and parallelly detect four typical pollutants in real water with high sensitivity and specificity was demonstrated. The limits of detection of MC-LR, 2,4-D, ATZ, and BPA were 0.04 µg/L, 0.09 µg/L, 0.02 µg/L, and 0.03 µg/L, respectively. The FMB could also achieve early-warning of pollutants thanks to its ability of rapidity, high-frequency, and multiple-analyte detection. The FMB has significant implications as a multiplexable, portable, rapid, and quantitative detection platform for pollution accidents and water quality management to satisfy the increasing demands of alerting and protecting civilians.