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Cell-free biosensors have inspired low-cost and field-applicable methods to detect antibiotic contaminants. However, the satisfactory sensitivity of current cell-free biosensors is mostly achieved by sacrificing the rapidity, which prolongs turnaround time by hours. Additionally, the software-based result interpretation provides an obstacle for delivering these biosensors to untrained individuals. Here, we present a bioluminescence-based cell-free biosensor, termed enhanced Bioluminescence sensing of Ligand-Unleashed RNA Expression (eBLUE). The eBLUE leveraged antibiotic-responsive transcription factors to regulate the transcription of RNA arrays that can serve as scaffolds for reassembling and activating multiple luciferase fragments. This process converted target recognition into an amplified bioluminescence response, enabling smartphone-based quantification of tetracycline and erythromycin directly in milk within 15 min. Moreover, the detection threshold of eBLUE can be easily tuned according to the maximum residue limits (MRLs) established by government agencies. Owing to this tunable nature, the eBLUE was further repurposed as an on-demand semi-quantification platform, allowing for fast (∼20 min) and software-free identification of safe and MRL-exceeding milk samples only by glancing over the smartphone photographs. Overall, the sensitivity, rapidity and user-friendliness of eBLUE demonstrate its potentials for practical applications, especially in resource-limited and household settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cytochrome P450 55B1(CYP55B1) from Chlamydomonas reinhardtii reduces nitric oxide (NO) to dinitrogen oxide (N 2 O) with the electron supply from NAD(P)H in vivo. Here a novel nitric oxide biosensor was developed by immobilized CYP55B1 on the surface of pyrolytic graphite electrode (PGE) by cross-linking with glutaraldehyde (GA) and bovine serum albumin (BSA). The direct electrochemistry of CYP55B1 was realized with the redox peak potential of -0.355 V and -0.385 V and the catalytic reduction peak of NO by CYP55B1 is at -0.85 V at the scan rate of 0.5 V S -1 in pH 7.0 phosphate buffer. The apparent coverage (Γ = 1.43 × 10 -11  mol cm -2 ), the electron transfer rate constant (ks = 17.39 s -1 ) and apparent affinity to NO (K mapp  = 11.64 nM) of CYP55B1 in GA/BSA film were obtained. The catalytic mechanism of CYP55B1 towards NO with NADH was examined by the biosensor. The linear range of NO detection was investigated by differential pulse voltammetry with the results of 5-50 nM and the detection limit of 0.5 nM (S/N = 3). The selectivity and stability of the electrochemical biosensor were investigated. Furthermore, the CYP55B1electrochemical biosensor was applied to monitor NO release from Arabidopsis protoplasts with the average content of 0.848 fmol per cell under anaerobic condition., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Detection of dysregulated circulating microRNAs (miRNAs) in human biofluids is a fundamental ability to determine tumor occurrence and metastasis in a minimally invasive fashion. However, the requirements for sophisticated instruments and professional personnel impede the translation of miRNA tests into routine clinical diagnostics, especially for resource-limited regions. Herein, we developed a DNA-guided bioluminescence strategy for the detection of circulating miRNAs. In this strategy, a pair of split luciferase-DNA chimeras was constructed and integrated into the miRNA-triggered rolling circle amplification (RCA) process. The tandem reassembly of split luciferase-DNA chimeras on the RCA products elicited a turn-on bioluminescence response with ultrahigh signal-to-background (S/B) ratio. This strategy enabled smartphone-based assays for different miRNAs with attomolar sensitivity and single-base specificity, as demonstrated here for miR-21. miR-148b, and cel-miR-39. Further application of our approach to the clinical serum samples realized identification of dysregulated miR-21 and miR-148b in the lung cancer patients, showing a satisfactory agreement with the control assays performed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Therefore, the developed method possesses the benefits of high performance and reliability, offering a potential tool for implementing miRNA-based diagnosis in point-of-care (POC) settings., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH 2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E 0' ) of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH 2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 μM and a lower limit of detection of 0.7 μM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins., (Copyright © 2019. Published by Elsevier B.V.)

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0.70 μmol/g of the total protein, which showed a single band on a SDS-PAGE and Western blot. The direct electrochemistry of CYP55B1 in dihexadecylphosphate (DHP) film was realized with an electric potential at -0.47 V at the scan rate of 1 V s -1 . We studied the in vitro interaction between P450 55B1 and NO by the fluorescence spectrometric method. The results show that the fluorescence intensity of iron-porphyrin in P450 55B1 changes gradually with the addition of NO. The fluorescence intensity change values against NO concentrations were plotted, and it showed a linear range of NO from 0 to 22.5 μM with a sensitivity of 0.15 μM/AU and a detection limit of 0.15 μM.

Ferric and ferrous ion plays critical roles in bioprocesses, their influences in many fields have not been fully explored due to the lack of methods for quantification of ferric and ferrous ions in biological system or complex matrix. In this study, an M13 bacteriophage (phage) was engineered for use as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein. The interaction between the specific phenol group of tyrosine and Fe(3+) / Fe(2+) was used as the sensor. Transmission electron microscopy showed aggregation of the tyrosine-displaying phages after incubation with Fe(3+) and Fe(2+). The aggregated phages infected the host bacterium inefficiently. This phenomenon could be utilized for detection of ferric and ferrous ions. For ferric ions, a calibration curve ranging from 200 nmol/L to 8 μmol/L with a detection limit of 58 nmol/L was acquired. For ferrous ions, a calibration curve ranging from 800 nmol/L to 8 μmol/L with a detection limit of 641.7 nmol/L was acquired. The assay was specific for Fe(3+) and Fe(2+) when tested against Ni(2+), Pb(2+), Zn(2+), Mn(2+), Co(2+), Ca(2+), Cu(2+), Cr(3+), Ba(2+), and K(+). The tyrosine displaying phage to Fe(3+) and Fe(2+) interaction would have plenty of room in application to biomaterials and bionanotechnology.

Using the plasmid pCW, high-level expression of rat cytochrome p4501A1 (CYP1A1) has been achieved by making NH(2)-terminal translational fusions to bacterial leader sequences ompA (ompA-1A1/pCW). The construct ompA-1A1 was compared with an expression construct in which the Ala codon GCT was placed in the second position and 5'-terminal codons were maximized for A T content (1A1/pCW). Both constructs produced spectrally active, functional protein. However, the ompA-1A1 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with the construct 1A1/pCW. The expressed 1A1 from the construct ompA-1A1/pCW in bacterial membrane fractions were collected and immobilized in nano-Na-montmorillonite (nano-SWy-2) and dihexadecylphosphate (DHP) composite film. The direct electrochemistry of CYP1A1 in a nano-SWy-2-DHP film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to benzo[a]pyrene has been investigated by the cyclic voltammetry. The immobilized CYP1A1 displayed a pair of redox peaks with a formal potential of -0.36 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 1 V s(-1). The CYP1A1 in the nano-SWy-2-DHP film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate benzo[a]pyrene (B[a]P) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP1A1 to B[a]P. By amperometry a calibration linear range for B[a]P was obtained to be 3.31-16.56 μM with a sensitivity of 58.57 μA mM(-1). And the apparent Michaelis-Menten constant for the electrocatalytic activity of CYP1A1 was estimated to be 46.27 μM for B[a]P., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Colloid Au (Au(nano)) with a diameter of about 10 nm was prepared and used in combination with dihexadecylphosphate (DHP) to immobilize glucose oxidase (GOD) onto the surface of a graphite electrode (GE). The direct electrochemistry of GOD confined in the composite film was investigated. The immobilized GOD displayed a pair of redox peaks with a formal potential of -0.475 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 150 mV s(-1). The GOD in the composite film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction peak current of dissolved oxygen decreased, which could be developed for glucose determination. A calibration linear range of glucose was 0.5-9.3 mM with a detection limit of 0.1 mM and a sensitivity of 1.14 microA mM(-1). The glucose biosensor showed good reproducibility and stability. The general interferences that coexisted in human serum sample such as ascorbic acid and uric acid did not affect glucose determination.

Colloid Au (Au(nano)) with a diameter of about 20 nm was prepared and used in combination with the multi-wall carbon nanotubes (MWNTs) to modify a gold electrode. Dihexadecylphosphate (DHP) dispersed in Au(nano) aqueous solution was used to solubilize MWNTs. Deposition of Au(nano) on MWNTs was realized as illustrated by TEM micrographs. The DHP formed a network that connected Au(nano) and MWNTs to the gold electrode surface. The Au(nano)-MWNTs-DHP composite film on the gold electrode surface was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammmetry (CV). The composite film modified gold electrode was used to detect cytochrome c and a pair of well-defined redox waves was obtained. It was found that the composite film promoted the redox of horse heart cytochrome c and its effect was developed for the determination of cytochrome c.

In this paper, we described a glucose biosensor based on the co-electrodeposition of a poly(vinylimidazole) complex of [Os(bpy)2Cl](+/2+) (PVI-Os) and glucose oxidase (GOX) on a gold electrode surface. The one-step co-electrodeposition method provided a better control on the sensor construction, especially when it was applied to microsensor construction. The modified electrode exhibited the classical features of a kinetically fast redox couple bound to an electrode surface and the redox potential of the redox polymer/enzyme film was 0.14 V (vs. SCE). For a scan rate of up to 200 mV s(-1), the peak-to-peak potential separation was less than 25 mV. In the presence of glucose, a typical catalytic oxidation current was observed, which reached a plateau at 0.25 V (vs. SCE). Under the optimal experimental conditions, the steady-state electrooxidation current measured at 0.30 V (vs. SCE) was linear to the glucose concentration in the range of 0-30 mM. Successful attempts were made in blood sample analysis.