Optimizing Microfluidic Impedance Cytometry by Bypass Electrode Layout Design.

Microfluidic impedance cytometry (MIC) has emerged as a popular technique for single-cell analysis. Traditional MIC electrode designs consist of a pair of (or three) working electrodes, and their detection performance needs further improvements for microorganisms. In this study, we designed an 8-electrode MIC device in which the center pair was defined as the working electrode, and the connection status of bypass electrodes could be changed. This allowed us to compare the performance of layouts with no bypasses and those with floating or grounding electrodes by simulation and experiment. The results of detecting Φ 5 µm beads revealed that both the grounding and the floating electrode outperformed the no bypass electrode, and the grounding electrode demonstrated the best signal-to-noise ratio (SNR), coefficient of variation (CV), and detection sensitivity. Furthermore, the effects of different bypass grounding areas (numbers of grounding electrodes) were investigated. Finally, particles passing at high horizontal positions can be detected, and Φ 1 µm beads can be measured in a wide channel (150 µm) using a fully grounding electrode, with the sensitivity of bead volume detection reaching 0.00097%. This provides a general MIC electrode optimization technology for detecting smaller particles, even macromolecular proteins, viruses, and exosomes in the future.

A disposable paper-based electrochemical biosensor decorated by electrospun cellulose acetate nanofibers for highly sensitive bio-detection.

Paper-based electrochemical sensors have the characteristics of flexibility, biocompatibility, environmental protection, low cost, wide availability, and hydropathy, which make them very suitable for the development and application of biological detection. This work proposes electrospun cellulose acetate nanofiber (CA NF)-decorated paper-based screen-printed (PBSP) electrode electrochemical sensors. The CA NFs were directly collected on the PBSP electrode through an electrospinning technique at an optimized voltage of 16 kV for 10 min. The sensor was functionalized with different bio-sensitive materials for detecting different targets, and its sensing capability was evaluated by CV, DPV, and chronoamperometry methods. The test results demonstrated that the CA NFs enhanced the detection sensitivity of the PBSP electrode, and the sensor showed good stability, repeatability, and specificity (p < 0.01, N = 3). The electrochemical sensing of the CA NF-decorated PBSP electrode exhibited a short detection duration of ∼5-7 min and detection ranges of 1 nmol mL-1-100 µmol mL-1, 100 fg mL-1-10 µg mL-1, and 1.5 × 102-106 CFU mL-1 and limits of detection of 0.71 nmol mL-1, 89.1 fg mL-1, and 30 CFU mL-1 for glucose, Ag85B protein, and E. coli O157:H7, respectively. These CA NF-decorated PBSP sensors can be used as a general electrochemical tool to detect, for example, organic substances, proteins, and bacteria, which are expected to achieve point-of-care testing of pathogenic microorganisms and have wide application prospects in biomedicine, clinical diagnosis, environmental monitoring, and food safety.

Direct, ultrafast, and sensitive detection of environmental pathogenic microorganisms based on a graphene biosensor.

Pathogenic microorganisms in the environment pose a serious threat to global human health. This study developed a reduced graphene oxide (rGO)-field effect transistor (FET) biosensor to realize the rapid and sensitive detection of pathogenic microorganisms. The rGO-FET sensors were prepared by in-situ thermal reduction method, and biorecognition elements were immobilized using a crosslinking agent to realize the surface functionalization of rGO. The rGO-FET biosensors can detect Escherichia coli O157:H7 as low as 1.4 CFU mL-1 within 46 s. The normalized current response was linearly correlated with E. coli concentration in the range of 1.4-1.4 × 107 CFU mL-1. The normalized current response of E. coli O157:H7 was about an order of magnitude higher than those of other microorganisms, indicating that the biosensor has good specificity. The current loss rates of the unmodified rGO-FET sensors and the biosensors modified with anti-E. coli O157:H7 after 30 days of storage at 4 °C were approximately 8% and 15%, respectively. Most importantly, the rGO-FET biosensors can directly detect real samples without pretreatment. Compared with other technologies, the rGO-FET biosensors can detect pathogenic microorganisms with a wider linear range in a shorter time, which is of great importance for the rapid warning and control of pathogenic microorganisms in the environment.

Rapid detection of airborne protein from Mycobacterium tuberculosis using a biosensor detection system.

Tuberculosis (TB), caused by infection with airborne Mycobacterium tuberculosis (MTB), seriously threatens human health and has become a public health problem of worldwide concern. To achieve effective control of TB, rapid and sensitive detection of MTB is particularly important. At present, the common detection methods for MTB cannot meet the requirements of speed, flexibility and portability simultaneously. In this work, a multichannel microfluidic chip was developed and packaged with an ultra-sensitive silicon nanowire field-effect-transistor biosensor. The fluid system was tested and optimized through simulation, and the best conditions were determined: the flow rate was 0.3 mL min-1 and the flow direction was perpendicular to a silicon nanowire. A one-way valve, a switching valve and a peristaltic pump were combined to establish a biosensor detection system to realize the automatic detection of TB samples. Then we systematically explained the factors affecting simulated exhaled breath condensate (SEBC) collection, and established and optimized the method for collection of SEBC from the perspective of collection volume and biological activity. The best collection conditions were determined for a 5 mm pipe diameter at 0 °C, and a sufficient sample volume was obtained in only 2 minutes for microfluidic detection. Then, the actual application value of the established collection method was further evaluated. Volunteers were recruited and this method was used to collect their exhaled breath condensate to analyze the collection effect. The system detected MTB in SEBC with good sensitivity (∼4 × 104 particles per mL). It is expected to be further integrated and miniaturized in the future to realize point-of-care testing.

Portable immunosensor directly and rapidly detects Mycobacterium tuberculosis in sputum.

Tuberculosis (TB) remains a public health problem that cannot be ignored. The portable and efficient detection of Mycobacterium tuberculosis (MTB) is important for the effective control of this disease. However, current detection techniques do not meet the requirements for MTB detection in the actual environment and often require cumbersome detection steps that are time consuming and inflexible. In this study, a portable immunosensor to detect MTB in sputum was prepared and then subjected to interface characterizations, such as scanning electron microscopy, hydrophilic angle test, and fluorescence characterization. The source and gate voltage of the device were optimized and tested using a non-contact photoresponse. The results showed that the sensitivity of the sensor to luminance increases with the decrease in source voltage. The gate voltage can substantially improve the response of the immunosensor to the normalized current of protein and amplify the signal at least 1.6 times. The optimal voltage detection conditions of source voltage (0.3 V) and gate voltage (0.1 V) were also determined. Several common proteins present in simulated saliva were used for anti-interference tests, and the sensor exhibited good specificity. Finally, the dilution gradient of an actual TB sputum sample was optimized. In the absence of preconditioning, a double-blind experiment was used to distinguish between the sputum from patients with TB and healthy individuals to shorten the TB detection time to a few minutes. Compared with the hospital's conventional detection method using cultures, the proposed method can complete the detection in a shorter time. This study provides a new strategy for the portable diagnosis of TB.

A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene editing is revolutionizing plant research and crop breeding. Here, we present an effective and streamlined pipeline for arrayed CRISPR library construction and demonstrate it is suitable for small- to large-scale genome editing in plants. This pipeline introduces artificial PCR fragment-length markers for distinguishing guide RNAs (gRNAs) (FLASH), and a group of 12 constructs harboring different FLASH tags are co-transformed into plants each time. The identities of gRNAs in Agrobacterium mixtures and transgenic plants can therefore be read out by detecting the FLASH tags, a process that requires only conventional PCR and gel electrophoresis rather than sequencing. We generated an arrayed CRISPR library targeting all 1,072 members of the receptor-like kinase (RLK) family in rice. One-shot transformation generated a mutant population that covers gRNAs targeting 955 RLKs, and 74.3% (710/955) of the target genes had three or more independent T0 lines. Our results indicate that the FLASH tags act as bona fide surrogates for the gRNAs and are tightly (92.1%) associated with frameshift mutations in the target genes. In addition, the FLASH pipeline allows for rapid identification of unintended editing events without corresponding T-DNA integrations and generates high-order mutants of closely related RLK genes. Furthermore, we showed that the RLK mutant library enables rapid discovery of defense-related RLK genes. This study introduces an effective pipeline for arrayed CRISPR library construction and provides genome-wide rice RLK mutant resources for functional genomics.

Rapid and Sensitive Detection of Mycobacterium tuberculosis by an Enhanced Nanobiosensor.

Tuberculosis (TB) mostly spreads from person to person through Mycobacterium tuberculosis (MTB). However, the majority of conventional detection methods for MTB cannot satisfy the requirements for actual TB detection. As one of the most promising powerful platforms, a silicon nanowire field-effect transistor (SiNW-FET) biosensor shows good prospect in TB detection. In this study, an enhanced SiNW-FET biosensor was developed for the rapid and sensitive detection of MTB. The surface functional parameters of the biosensor were explored and optimized. The SiNW-FET biosensor has good sensitivity with a detection limit of 0.01 fg/mL toward protein. The current change value shows a linear upward trend with the increase in protein concentration in the range of 1 fg/mL to 100 µg/mL. One whole test cycle can be accomplished within only 30 s. More importantly, a good distinction was realized in the sputum without pretreatment between normal people and TB patients, which greatly shortened the TB detection time (only 2-5 min, considering the dilution of sputum). Compared with other methods, the SiNW-FET biosensor can detect MTB with a remarkably broad dynamic linear range in a shorter time.

Advances in airborne microorganisms detection using biosensors: A critical review.

Humanity has been facing the threat of a variety of infectious diseases. Airborne microorganisms can cause airborne infectious diseases, which spread rapidly and extensively, causing huge losses to human society on a global scale. In recent years, the detection technology for airborne microorganisms has developed rapidly; it can be roughly divided into biochemical, immune, and molecular technologies. However, these technologies still have some shortcomings; they are time-consuming and have low sensitivity and poor stability. Most of them need to be used in the ideal environment of a laboratory, which limits their applications. A biosensor is a device that converts biological signals into detectable signals. As an interdisciplinary field, biosensors have successfully introduced a variety of technologies for bio-detection. Given their fast analysis speed, high sensitivity, good portability, strong specificity, and low cost, biosensors have been widely used in environmental monitoring, medical research, food and agricultural safety, military medicine and other fields. In recent years, the performance of biosensors has greatly improved, becoming a promising technology for airborne microorganism detection. This review introduces the detection principle of biosensors from the three aspects of component identification, energy conversion principle, and signal amplification. It also summarizes its research and application in airborne microorganism detection. The new progress and future development trend of the biosensor detection of airborne microorganisms are analyzed.

A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants.

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.

Simultaneously enhancing degradation of refractory organics and achieving nitrogen removal by coupling denitrifying biocathode with MnOx/Ti anode.

To simultaneously remove carbon and nitrogen from refractory organic wastewater, this study couples the denitrifying biocathode and MnOx/Ti anode to oxidize refractory organic pollutants in the anode chamber and remove NO3--N in the cathode chamber (denitrifying biocathode-electrocatalytic reactor, DBECR). After inoculation, DBECR started up at 1.3 and 1.5 V with NO3--N reduction peak appearing on the cyclic voltammetry curve and increased NO3--N removal by approximately 90 %. Compared to the electrocatalytic reactor without inoculation (ECR), NO3--N removal of DBECR significantly increased from 0.09 to 0.45 kg NO3--N/m3 NCC/d (NCC: net cathodic compartment). NO3--N removal correlated well with charges/current flowing through the circuit of DBECR, further validating the presence of electrotrophic denitrifiers. Moreover, coupling of denitrifying biocathode significantly enhanced methylene blue (MB) removal in the anode chamber (0.18 ± 0.002 and 2.92 ± 0.02 g COD/m2/d for ECR and DBECR, respectively). This was because the growth of eletrotrophic denitrifiers increased the cathodic potential and thus the potential of MnOx/Ti anode. The higher potential of MnOx/Ti anode promoted the generation of hydroxyl radicals and consequently promoted MB removal. This study demonstrated that DBECR not only realized nitrogen removal in the cathode chamber, but also enhanced refractory organic carbon degradation in the anode chamber.

Metabolic Functional Community Diversity of Associated Bacteria during the Degradation of Phytoplankton from a Drinking Water Reservoir.

In the drinking water reservoir ecosystem, phytoplankton and bacteria play important roles in shaping freshwater health and function. In this work, the associated bacterial community functional diversity during degradation of phytoplankton was determined using the substrate utilization profiling (BIOLOG) technique, meanwhile, the composition and concentration of phytoplankton were examined using a microscope. The results indicated that Euglena decreased 58.33% from 0 to 38 d, while the smallest degradation of Bacillariophyta was 20.19%. Average well color development (AWCD590nm) increased during the static periods from 0 to 38 d; however, the AWCD590nm of 18 and 38 d had no significant difference (p ＜ 0.05). The Simpson's index (D) was in accordance with Shannon's diversity (H) and species richness(S); it was measured to be18 > 38 > 5 > 0d. There were significant differences in the pattern and level of carbon sources used by the phytoplankton-associated bacteria. In addition, the principle component analyses (PCA) suggested that the first principle component (PC1) and the second principle component (PC2) explained 46.76% and 21.49% of the total variation for bacterial community, respectively. Redundancy analysis (RDA) revealed that cell abundance of phytoplankton was negatively correlated with the AWCD590nm, amino acids and other functional indexes. Therefore, the data suggest that there are differences in the phytoplankton-associated bacterial community functional diversity during different static stages of water samples collected from the drinking water reservoir.

Enhanced anodic oxidation and energy saving for dye removal by integrating O2-reducing biocathode into electrocatalytic reactor.

Simultaneous enhancement of dye removal and reduction of energy consumption is critical for electrochemical oxidation in treating dyeing wastewater. To address this issue, this work presented a novel process termed biocathode-electrocatalytic reactor (BECR). The dual-chamber BECR employed O2-reducing biocathode instead of normal stainless steel (SS) cathode and MnOx/Ti anode to reduce O2 in the cathode chamber and treat methylene blue (MB) in the anode chamber, respectively. BECR successfully started up at 0.7 and 1 V and substantially improved MB and total organic carbon (TOC) removal compared with the electrocatalytic reactor with SS cathode (ECR-SS), e.g., removal of MB (150 mg L-1) increased from 27.0 ± 0.2% to 78.1 ± 0.4% at 1 V. To achieve the same TOC removal, BECR reduced the energy consumption by approximately 45.7% compared with ECR-SS (19.5 and 35.9 kWh (kg TOC) -1 for BECR and ECR, respectively). To explain the above merits of BECR, M(·OH) (·OH adsorbed on the anode surface) generation, potential of MnOx/Ti anode (Ea), and their correlation were investigated. When coupled with O2-reducing biocathode, MnOx/Ti anode considerably accelerated M(·OH) generation because Ea increased. The increased Ea in BECR was due to the fact that its cathodic reaction was converted to the four-electron O2 reduction, which exhibited a higher cathodic potential than hydrogen evolution reaction on SS cathode in ECR-SS. Thereby, BECR simultaneously promoted dye removal and reduced energy consumption, showing promise in treating dyeing wastewater.