Rapid and sensitive parallel on-site detection of antibiotics and resistance genes in aquatic environments using evanescent wave dual-color fluorescence fiber-embedded optofluidic nanochip.

Environmental antibiotics and antibiotic resistance genes (ARGs) pose considerable threat to humans and animals; thus, the rapid and sensitive parallel detection of these pollutants from a single sample is urgently required. However, traditional multiplexed analytic technologies detect only one type of target (e.g., small molecules or nucleic acids) per assay. To address this issue, Evanescent wave Dual-color fluorescence Fiber-embedded Optofluidic Nanochip (EDFON) was fabricated by integrating a fiber-embedded optofluidic nanochip with evanescent wave dual-color fluorescence technology. The EDFON was used for the parallel quantitative detection of sulfamerazine (SMR) and MCR-1 with high sensitivity and specificity by combining a heterogeneous immunoassay with a homogenous hybridization chain reaction based on time-resolved effects. LODs of 0.032 µg/L and 35 pM was obtained for SMR and MCR-1, respectively, within 20 min. To our best knowledge, the EDFON is the first device for the simultaneous detection of two type of targets in each test, which is highly valuable to prevent the global threats of antibiotics and ARGs. Comparison with liquid chromatography-mass spectrometry showed a strong linear relationship (R2 = 0.998) for SMR pollution in the Qinghe River, with spiked SMR and MCR-1 negative surface and wastewater samples showing recovery rates of 91.8-113.4%. These results demonstrate the excellent accuracy and reliability of the EDFON, with features such as multi-analyte detection, field-deployment, and minimal-equipment, rendering it revolutionary for environmental monitoring, food safety, and medical diagnostics.

Rapid and sensitive point-of-need aflatoxin B1 testing in feedstuffs using a smartphone-powered mobile microfluidic lab-on-fiber device.

Rapid, high-frequency, and accurate identification of aflatoxin B1 (AFB1) is crucial for ensuring food safety and reducing population mortality. Herein, we constructed Smartphone powered Mobile mIcrofluidic Lab-on-fiber dEvice (SMILE) comprising a compact optical system, fiber nano-bioprobe-embedded microfluidic-chip system, mini-photodetector, and software application to facilitate the rapid and sensitive point-of-need quantitative testing for AFB1. The elegant optical design of SMILE significantly improves light transmission efficiency, detection sensitivity, and portability by integrating a compacted all-fiber optical structure with a fiber nano-bioprobe-embedded microfluidic chip. Furthermore, the nanopore layer of the fiber nano-bioprobe improves detection sensitivity by increasing the biorecognition molecule number and enhancing the interaction between the evanescent field and dye. Through an indirect competitive immunoassay mechanism, SMILE achieves sensitive quantitative detection of AFB1 with a detection limit of 0.08 µg/L. Herein, SMILE was validated using several feedstuff samples tested with a simple aqueous extraction protocol, demonstrating good correlation with high-performance liquid chromatography for AFB1-contaminated feedstuffs. The immunoassay process is completed within 12 min, boasting high sensitivity, specificity, reusability, and reproducibility. Owing to its sensitivity, portability, flexibility, plug-and-play, and smartphone integration, SMILE is highly scalable for rapid and high-frequency point-of-need testing for AFB1 and other trace contaminants.

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.

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.

Reusable smartphone-facilitated mobile fluorescence biosensor for rapid and sensitive on-site quantitative detection of trace pollutants.

Increasing exposure to toxic pollutants highlights the need for their sensitive detection technologies that can be rapidly adapted and deployed in various settings. Optical biosensors are an excellent solution due to their outstanding features. However, the sophisticated and expensive optical design limits their scalability and actual application. Herein, an innovative reusable smartphone-facilitated mobile fluorescence biosensor (s-MFB) was built through integrating miniaturized all-fiber optical system and microfluidic system with smartphone. An asymmetric Y-shaped fiber optic coupler (Y-FOC) is constructed for simultaneous transmission of excitation light and the collected fluorescence. In particular, the incidence rays are introduced into the fiber bio-probe at a specific angle through the single-mode fiber of the Y-FOC, which enhances the evanescent wave field and the number of total internal reflections. The s-MBF showed a LOD for free Cy5.5 of 0.1 nM. Combining indirect competitive immunoassay with the s-MFB, this new assay, which achieve quantitative detection of bisphenol A and norfloxacin in 15 min with high sensitivity and reusability, substantially reduces the complexity and improves the scalability of trace pollutants detection. The adjunctive smartphone application allows on-site real-time quantitative detection, automated interpretation of reporting results, and early-warning of pollution accidents.

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.

Simultaneous and sensitive determination of Escherichia coli O157:H7 and Salmonella Typhimurium using evanescent wave dual-color fluorescence aptasensor based on micro/nano size effect.

The simultaneous and sensitive determination of two common pathogenic bacteria, Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) was achieved using evanescent wave dual-color fluorescence aptasensor and the fiber nanoprobe through combining the micro/nano size and time-resolved effect. Two fluorescence labeled aptasensors, Cy3-apt-E and Cy5.5-apt-S, were regarded as biorecognition elements and signal reporters for E. coli O157:H7 and S. Typhimurium, which were alternatively excited by evanescent waves originated from 520 nm to 635 nm excitation lights, respectively. The fiber nanoprobe with in-situ etched nanopores was used for distinguishing free aptasensors and aptasensors bound to pathogenic bacteria based on the limited penetrated depth of evanescent wave and the significant size difference of bacteria and nanopore. The E. coli O157:H7 and S. Typhimurium were directly and simultaneously quantitated in less than 35 min without the requirement of the complex immobilization of biorecognition molecules and bacteria enrichment/separation processes. The limits of detection of E. coli O157:H7 and S. Typhimurium were 340 CFU/mL and 180 CFU/mL, respectively. The satisfied recovery rate of real samples testing verified the feasibility and accuracy of the proposed method. Our strategy not only greatly simplifies the detection and identification process of multiple pathogenic bacteria, but also is easy to extend as a universal technology for sensitive determination of other bacteria using their respective biorecognition molecules.