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As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced "one to many" FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis.

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Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 µM or KD = 1.6 µM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

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Endotoxins, also known as lipopolysaccharides (LPS), are present within the cell walls of Gram-negative bacteria and are released upon cellular death, which can pose a significant risk to human and animal health. Due to the minimal amount of endotoxin required to trigger an inflammatory response in human body, the demand for sensitive methods with low endotoxin detection limits is essential necessary. This paper presents a straightforward aptamer sensor which can enhance the conductivity and specific surface area of molybdenum disulfide (MoS2) by incorporating carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and polyaniline (PANI). Doping with gold nanoparticles (AuNPs) improves biocompatibility and sensitivity while providing binding sites for thiolated endotoxin-binding aptamers (LBA). This biosensor achieved a remarkable detection limit as low as 0.5 fg mL-1, enabling trace-level identification of LPS. It also exhibits excellent repeatability, selectivity, and stability, facilitating rapid and accurate LPS detection. Moreover, this method demonstrates high recovery rates and specificity for LPS analysis in food samples, showcasing its promising application prospects in trace-level LPS detection within the food industry.

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Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant "xenonucleic acid" 2'O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2'O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2'O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2'O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.

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An electrochemiluminescence (ECL) sensing platform for ultrasensitive and highly selective detection of kanamycin (KANA) was developed based on the prepared Ru(bpy)32+-functionalized MOF (Ru@MOF) composites by hydrothermal synthesis and Ag+-dependent DNAzyme. In this sensor, the stem-loop DNA (HP) with the ferrocene (Fc) was used as substrate chain to quench the ECL emission generated by the Ru@MOF. Using the specific recognition effect between KANA and the KANA aptamer (Apt) and the DNAzyme dependence on Ag+, the KANA aptamer as the pendant strand of the DNAzyme was assembled on Ru@MOF/GCE with the aptamer. When both Ag+ and KANA were present simultaneously, KANA specifically was binded to KANA aptamer as a pendant chain. Subsequently, Ag+-dependent DNAzyme walker continuously cleaved the HP chain and released the modified end of Fc to restore the ECL signal of Ru@MOF composites, thus achieving selective and ultrasensitive detection of KANA. The constructed KANA biosensor exhibits a wide detection range (30 pM to 300 µM) accompanied by a low detection limit (13.7 pM). The KANA in seawater and milk samples are determined to evalute the practical application results of the sensor. This ECL detection strategy could be used for detecting other similar analytes and has broad potential application in biological analysis.

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A highly sensitive tumor necrosis factor α (TNF-α) detection method based on a surface-enhanced Raman scattering (SERS) magnetic patch sensor is reported. Magnetic beads (MNPs) and core shells were used as the capture matrix and signaling probe, respectively. For this purpose, antibodies were immobilized on the surface of magnetic beads, and then Au@4-MBN@Ag core-shell structures coupled with aptamers and TNF-α antigen were added sequentially to form a sandwich immune complex. Quantitative analysis was performed by monitoring changes in the characteristic SERS signal intensity of the Raman reporter molecule 4-MBN. The results showed that the limit of detection (LOD) of the proposed method was 4.37 × 10-15 mg·mL-1 with good linearity (R2 = 0.9918) over the concentration range 10-12 to 10-5 mg·mL-1. Excellent assay accuracy was also demonstrated, with recoveries in the range 102% to 114%. Since all reactions occur in solution and are separated by magnetic adsorption of magnetic beads, this SERS-based immunoassay technique solves the kinetic problems of limited diffusion and difficult separation on solid substrates. The method is therefore expected to be a good clinical tool for the diagnosis of the inflammatory biomarker THF-α and in vivo inflammation screening.

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A simple, fast, low cost, sensitive, intuitive, visual, label-free, and smartphone-assisted aptamer sensor based on colorimetric assay for the measurement of zearalenone was constructed. The nucleic acid aptamer of zearalenone was used as the recognition element and gold nanoparticles were used as the indicator. Several factors that could influence sensitivity, including the concentration of aptamer and NaCl, and incubation time, and specificity, have been investigated. The results showed that under the optimal conditions, the signal had a good linear relationship when zearalenone concentration is 5-300 ng/mL. A linear regression equation is Y = 0.0003X + 0.5128 (R2 = 0.9989) and a limit of detection is 5 ng/mL. The specificity of the sensor was good. Zearalenone in maize samples were successfully measured. The recoveries of Zearalenone are 81.3 %-96.4 %. The whole process takes only 15 min to complete. The smartphone assisted colorimetric aptamer sensor can be used for the detection of zearalenone in maize.

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The labeling-free and immobilization-free homogeneous aptamer sensor offers advantages including simple operation, low cost, and high sensitivity, demonstrating great potential in rapid detection of tumor biomarkers in biological samples. In this work, a labeling-free and immobilization-free homogeneous aptamer sensor was conveniently fabricated by combining size exclusion and charge-selective penetration of a nanochannel-modified electrode and two-dimensional (2D) nanorecognition probe which can realize selective and highly sensitive detection of alpha-fetoprotein (AFP) in serum. Vertically ordered mesoporous silica film (VMSF) with ultra-small, uniform, and vertically aligned nanochannels was easily grown on the simple, low-cost, and disposable indium tin oxide (ITO) electrode. Through π-π interaction and electrostatic force, the AFP aptamer (Apt) and electrochemical probe, tris(bipyridine)ruthenium(II) (Ru(bpy)32+), were coloaded onto graphene oxide (GO) through simple incubation, forming a 2D nanoscale recognition probe (Ru(bpy)32+/Apt@GO). Owing to the size exclusion effect of VMSF towards the 2D nanoscale probe, the electrochemical signal of Ru(bpy)32+/Apt@GO could not be detected. In the presence of AFP, the specific binding of AFP to the aptamer causes the dissociation of the aptamer and Ru(bpy)32+ from GO, resulting in their presence in the solution. The efficient electrostatic enrichment towards Ru(bpy)32+ by negatively charged VMSF allows for high electrochemical signals of free Ru(bpy)32+ in the solution. Linear determination of AFP ranged from 1 pg/mL to 1000 ng/mL and could be obtained with a low limit of detection (LOD, 0.8 pg/mL). The high specificity of the adapter endowed the constructed sensor with high selectivity. The fabricated probe can be applied in direct determination of AFP in serum.

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Electrochemical aptamer-based (EAB) sensors are capable of measuring the concentrations of specific molecules in vivo, in real time, and with a few-second time resolution. For their signal transduction mechanism, these sensors utilize a binding-induced conformational change in their target-recognizing, redox-reporter-modified aptamer to alter the rate of electron transfer between the reporter and the supporting electrode. While a variety of voltammetric techniques have been used to monitor this change in kinetics, they suffer from various drawbacks, including time resolution limited to several seconds and sensor-to-sensor variation that requires calibration to remove. Here, however, we show that the use of fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate EAB sensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. To showcase these benefits, we demonstrate the approach's ability to perform real-time molecular measurements in the veins of living rats.

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A rapid and convenient homogeneous aptamer sensor with high sensitivity is highly desirable for the electrochemical detection of tumor biomarkers. In this work, a homogeneous electrochemical aptamer sensor is demonstrated based on a two-dimensional (2D) nanocomposite probe and nanochannel modified electrode, which can realize sensitive detection of carcinoembryonic antigen (CEA). Using π-π stacking and electrostatic interaction, CEA aptamer (Apt) and cationic redox probe (hexaammineruthenium(III), Ru(NH3)63+) are co-loaded on graphite oxide (GO), leading to a 2D nanocomposite probe (Ru(NH3)63+/Apt@GO). Vertically ordered mesoporous silica-nanochannel film (VMSF) is easily grown on the supporting indium tin oxide (ITO) electrode (VMSF/ITO) using the electrochemical assisted self-assembly (EASA) method within 10 s. The ultrasmall nanochannels of VMSF exhibits electrostatic enrichment towards Ru(NH3)63+ and size exclusion towards 2D material. When CEA is added in the Ru(NH3)63+/Apt@GO solution, DNA aptamer recognizes and binds to CEA and Ru(NH3)63+ releases to the solution, which can be enriched and detected by VMSF/ITO electrodes. Based on this mechanism, CEA can be an electrochemical detection ranging from 60 fg/mL to 100 ng/mL with a limit of detection (LOD) of 14 fg/mL. Detection of CEA in human serum is also realized. The constructed homogeneous detection system does not require the fixation of a recognitive aptamer on the electrode surface or magnetic separation before detection, demonstrating potential applications in rapid, convenient and sensitive electrochemical sensing of tumor biomarkers.

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Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L-1 to µmol L-1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

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A simple, rapid, low-cost, sensitive, intuitive, visual, label-free, colorimetric smartphone-assisted assay was developed for the measurement of aflatoxin B1 in miscellaneous beans. Ten different kinds of miscellaneous beans were treated and measured by modified QuEChERS(Quick、Easy、Cheap、Effective、Rugged、Safe) method with aflatoxin B1 nucleic acid aptamer as a recognition element and gold nanoparticles as indicators. Several factors influencing its sensitivity were investigated, including consumes and NaCl concentrations, as well as incubation time and specificity. The results showed a good linear relationship between concentrations of 0.2-8.0 ng/g under optimal conditions. With a detection limit of 0.08 ng/g, the linear regression equation was Y = 0.024X + 0.4615 (R = 0.9989). Sensor specificity is good. The content of aflatoxin B1 in bean samples was determined successfully. The recovery of aflatoxin B1 ranged from 87.18% to 110.24%. The whole thing took 15 min. This smartphone-assisted colorimetric aptamer sensor can be used to detect aflatoxin B1 in beans.

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A one-step electrodeposition-assisted self-assembly technique has been developed for preparation of ZnTCPP@MOF films with three-dimensional mesoporous structure in a three-electrode system. The internal structure of the ZnTCPP@MOF films was tuned by adjusting the electrochemical deposition voltage, deposition time, and the concentration of ZnTCPP at room temperature. The ZnTCPP@MOF films under different deposition conditions were characterized by scanning electron microscopy, Fourier transformation infrared spectroscopy, and X-ray photoelectron spectroscopy. The prepared ZnTCPP@MOF films exhibited excellent fluorescence properties, in which ZnTCPP molecules were encapsulated inside the MOF as fluorescent signal probes and structure-directing agents, which affected the electrochemical response of the ZnTCPP@MOF films. The sensing platform based on ZnTCPP@MOF film was used to detect microcystin with a wide determination range (1.0 × 10-12 mol/L ~ 1.0 × 10-5 mol/L), low determination limit (3.8 × 10-13 mol/L), and high sensitivity. More importantly, the strategy is simple, low-cost, green, and environmentally friendly, and it provides a new strategy for the direct use of MOFs films as signaling components.

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Evaluation of human epidermal growth factor receptor 2 (HER2) molecular markers is a very suitable option for early diagnosis of breast cancer. Metal-organic frameworks (MOFs) have large porosity and surface interactions such as π-π stacking, electrostatics, hydrogen bonding, and coordination. Here, we integrated the HER2 aptamer and fluorescent probe coumarin (COU) with zeolite imidazolic acid framework-8 (ZIF-8) to construct a label-free fluorescent aptamer sensor with pH-gated release of COU. In the presence of the target-HER2, the aptamer adsorbed on the surface of ZIF-8@COU specifically recognizes and falls off the HER2 protein, exposing a portion of the pore size of ZIF-8@COU while reducing the negative charge on the sensor surface, under alkaline hydrolysis conditions, a large number of COU fluorescent molecules can be produced and released in the detection system.The aptamer fluorescence sensor has good detection performance, sensitivity and low background interference, the detection linearity range of HER2 protein is 0.05-10 ng/mL, the detection limit is 0.0005 ng/mL, and it has good recovery rate for the serum detection of clinical breast cancer patients. Therefore, this sensor has high potential in detecting and monitoring HER2 levels for the care and clinical diagnosis of breast cancer patients.

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Amplified ATP imaging in inflammatory cells is highly desirable. However, the spatial selectivity of current amplification methods is limited, that is, signal amplification is performed systemically and not in a disease site-specific manner. Here we present a versatile strategy, termed enzymatically triggerable, aptamer-based signal amplification (ETA-SA), that enables inflammatory cell-specific imaging of ATP through spatially-resolved signal amplification. The ETA-SA leverages a translocated enzyme in inflammatory cells to activate DNA aptamer probes and further drive cascade reactions through the consumption of hairpin fuels, which, however, exerts no ATP response activity in normal cells, leading to a significantly improved sensitivity and spatial specificity for the inflammation-specific ATP imaging in vivo. Benefiting from the improved spatial selectivity, enhanced signal-to-background ratios were achieved for ATP imaging during acute hepatitis.

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Carcinoembryonic antigen (CEA) is a tumor-specific biomarker; however, its low levels in the early stages of cancer make it difficult to detect. To address the need for analysis of ultra-low-level substances, we designed and synthesized a fluorescent aptamer sensor with DNAzyme signal amplification and used it for the detection of CEA in blood. In the presence of the target protein, the aptamer sequence in the recognition probe binds to the target protein and opens the hairpin structure, hybridizes with the primer and triggers a polymerization reaction in the presence of polymerase to generate double-stranded DNA with two restriction endonuclease Nb.BbvCl cleavage sites. At the same time, the target protein is displaced and continues to bind to another recognition probe, triggering a new round of polymerization reaction, forming a cyclic signal amplification triggered by the target. The experimental results show that the blood detection with CEA has a high sensitivity and a wide detection range. The detection range: 10 fg/mL~10 ng/mL, with a detection limit of 5.2 fg/mL. In addition, the sensor can be used for the analysis of complex biological samples such as blood.

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In this paper, an electrochemiluminescence (ECL) biosensor was constructed using Bi2S3@Au nanoflowers as the based nanomaterial and Au@luminol and CdS QDs as independent ECL emission signal respectively. As the substrate of the working electrode, Bi2S3@Au nanoflowers improved the effective area of electrode and accelerated electron transfer rate between gold nanoparticles and aptamer, provided a good interface environment for the loading of luminescent materials. Then, the Au@luminol functionalized DNA2 probe was used as an independent ECL signal source under positive potential and recognized Cd(II), while the CdS QDs functionalized DNA3 probe was used as an independent ECL signal source under negative potential and recognized ampicillin. The simultaneous detection of Cd(II) and ampicillin in different concentrations are realized. This sensor not only has good selectivity and high sensitivity in real sample detection, but also open up a novel way to construct multi-target ECL biosensor for simultaneous detection.

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Vascular endothelial growth factor (VEGF) plays an important role in atherosclerosis, and the detection of VEGF is critical for the prevention, monitoring, and diagnosis of cardiovascular diseases. Here, a novel "signal on-off-super on" sandwich-type aptamer sensor with a triple signal amplification strategy was developed for the first time. Based on the capture aptamer was labeled with methylene blue (MB) on the internal bases, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-coupled voltage enrichment was used to amplify the electrochemical signal. To improve the analytical performance of the aptamer sensor, gold nanoparticles@Ti3C2Tx-Mxene (AuNPs@Ti3C2Tx-Mxene) were synthesized through the electrodeposition of AuNPs on the Ti3C2Tx-Mxene surface, providing active sites for the immobilization of the aptamer and amplifying the electrochemical signals. The excellent trans-cleavage activity of the CRISPR-Cas12a system was harnessed to cleave signal probes. The cleaved signal probes were enriched using an electrochemical signal instead of complicated target amplification steps before detection. Hence, we report a simplified detection process for amplifying electrochemical signals. Under optimal conditions, the aptamer sensor exhibited high sensitivity, acceptable stability, and reproducibility with a wide linear range from 1 pM to 10 µM (R2 = 0.9917) and an ultralow detection limit of 0.33 pM (S/N = 3). Therefore, we propose a novel strategy of CRISPR-Cas12a-based protein detection that opens a new window for the diagnostic applications of various biomarkers.

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Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd2+. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd2+-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd2+ and is able to measure the Cd2+ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd2+ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

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Exploiting a simple and sensitive sensor to efficiently detect streptomycin (STR) in milk is critical for resolving the harm caused to humans by STR residues. This study reports an electrochemical sensor using magnetic mesoporous carbon materials (MMCM) as a loaded material through magnetic adsorption immobilized on magnetic glassy carbon electrode (MGCE) and adsorbing unlabeled streptomycin aptamer (STP) as the identification element. The sensor can detect STR sensitively with a wide detection range (0.172-17.2 × 103 and 17.2 × 103 -17.2 × 105 nM) and a low detection limit of 0.015 nM. Experimental results revealed that the specific binding of STP with STR on the electrode changed the configuration of STP, thereby causing current change of differential pulse voltammetry curve. Compared with HPLC, this study provides a new method for rapid and sensitive detection of STR in milk (n = 5, 95 % confidence level, RSD＜5%).

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