Detection Of Target Molecules Research Articles

Zwitterionic Polymers Coating Antibiofouling Photoelectrochemical Aptasensor for In Vivo Antibiotic Metabolism Monitoring and Tracking.

Long-term in vivo monitoring and tracking of target molecules in living organism is essential to reveal vital physiological activity. However, undesirable contamination of protein and biological cells may bring serious biofouling issues. Herein, zwitterionic sulfobetaine methacrylate (SBMA) polymers are grafted on the TiO2 nanotube (NT) surface with polydopamine (PDA) as linker to fabricate a TiO2 NTs/PDA/SBMA photoelectrode. The TiO2 NTs/PDA/SBMA/aptamer-based PEC aptasensor can be sensitive and have selective detection of target molecules with excellent antibiofouling activity. Beneficial from the above advantages, the implantable micro-PEC aptasensor has implemented in vivo tracking and monitoring of the metabolism of antibiotics in a living mouse. The robust antibiofouling property generates new inquiries and an approach for long-standing questions in a new way for reliable and long-term sensing of vital biomolecules in complex biological fluids and uncovers a promising advance of intrinsic physiological mechanisms.

Screening Prostate Cancer Cell‐Derived Exosomal MicroRNA Expression with Photothermal‐Driven Digital PCR

AbstractExosomes are cell‐derived extracellular vesicles which enclose genetic substances from their origin cells, offering a good source of biomarkers. Many efforts have been made to develop non‐invasive exosome‐based clinical diagnosis, which are urgently needed for prostate cancer, as traditional methods are criticized due to the inaccuracy and discomfort. In this study, a simple, universal process for genotyping of exosome‐derived microRNAs using nanoparticles to capture exosomes and the novel photothermal droplet digital polymerase chain reaction (ddPCR) assay to detect target molecules is presented. To detect the target sensitively and simply, the smart ddPCR thermocycling of the agarose microcarriers can be controllably realized via embedding photothermal MXene and magnetic nanoparticles composite, and then irradiating with a homemade automated near‐infrared control module. Duplex photothermal‐driven ddPCR cooperating with dual TaqMan probes enabled simultaneously and quantitatively detecting copy numbers of prostate oncogenes microRNA‐21‐5p, microRNA‐375‐3p, and metastasis microRNA‐574‐3p along with the control microRNA‐30a‐5p within the extracted exosomes. The results show that the expression levels of these biomarkers in prostate cancer cell lines PC‐3 and DU145 are 7–210 folds higher than those in healthy control cells HaCaT and HUVEC. Thus, it is believed that these versatile exosomal microRNA genotyping approaches are potentially valuable for diverse clinical applications.

Spectral-Free Double Light Detection of DNA Based on a Porous Silicon Bragg Mirror.

To improve the detection sensitivity of a porous silicon optical biosensor in the real-time detection of biomolecules, a non-spectral porous silicon optical biosensor technology, based on dual-signal light detection, is proposed. Double-light detection is a combination of refractive index change detection and fluorescence change detection. It uses quantum dots to label probe molecules to detect target molecules. In the double-signal-light detection method, the first detection-signal light is the detection light that is reflected from the surface of the porous silicon Bragg mirror. The wavelength of the detection light is the same as the wavelength of the photonic band gap edge of the porous silicon Bragg mirror. CdSe/ZnS quantum dots are used to label the probe DNA and hybridize it with the target DNA molecules in the pores of porous silicon to improve its effective refractive index and enhance the detection-reflection light. The second detection-signal light is fluorescence, which is generated by the quantum dots in the reactant that are excited by light of a certain wavelength. The Bragg mirror structure further enhances the fluorescence signal. A digital microscope is used to simultaneously receive the digital image of two kinds of signal light superimposed on the surface of porous silicon, and the corresponding algorithm is used to calculate the change in the average grey value before and after the hybridization reaction to calculate the concentration of the DNA molecules. The detection limit of the DNA molecules was 0.42 pM. This method can not only detect target DNA by hybridization, but also detect antigen by immune reaction or parallel biochip detection for a porous silicon biosensor.

A Critical Review of the Use of Graphene-Based Gas Sensors

The employment of graphene for multifunctional uses has been a cornerstone in sensing technology. Due to its excellent electrochemical properties, graphene has been used in its pure and composite forms to detect target molecules over a wide range of surfaces. The adsorption process on the graphene-based sensors has been studied in terms of the change in resistance and capacitance values for various industrial and environmental applications. This paper highlights the performance of graphene-based sensors for detecting different kinds of domestic and industrial gases. These graphene-based gas sensors have achieved enhanced output in terms of sensitivity and working range due to specific experimental parameters, such as elevated temperature, presence of particular gas-specific layers and integration with specific nanomaterials that assist with the adsorption of gases. The presented research work has been classified based on the physical nature of graphene used in conjugation with other processed materials. The detection of five different types of gases, including carbon dioxide (CO2), ammonia (NH3), hydrogen sulphide (H2S), nitrogen dioxide (NO2) and ethanol (C2H5OH) has been shown in the paper. The challenges of the current graphene-based gas sensors and their possible remedies have also been showcased in the paper.

CRISPR/Cas12a-derived sensitive electrochemical biosensing of NF-κB p50 based on hybridization chain reaction and DNA hydrogel

Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.

Quantitative and Noninvasive Detection of SAH-Related MiRNA in Cerebrospinal Fluids In Vivo Using SERS Sensors Based on Acupuncture-Based Technology.

Quantitative analysis of microRNAs (miRNAs) in a noninvasive manner is of vital importance for disease diagnosis and prognosis evaluation. However, conventional strategies for realizing accurate, simple, and sensitive detection of target molecules are still a challenge, especially for miRNAs due to their low abundance and susceptibility in the complex biological environment. Here, a novel surface-enhanced Raman scattering (SERS) strategy was established for quantitative detection and monitoring of miRNA-21-5p (miR-21-5p) in living cells and in vivo cerebrospinal fluid (CSF) by applying hairpin DNA (hpDNA)-conjugated gold nanostars (GNSs) SERS probes combined with acupuncture-based technology. This strategy enabled ultrasensitive exploration toward miR-21-5p in a wide range from 1 fM to 100 pM in cell lysates. Moreover, SERS analysis facilitated the detection and long-term monitoring for in vivo miR-21-5p noninvasively. This developed strategy promises to offer a powerful method for the analysis of multiple biomolecules in single cells and living bodies.

Sensitive detection of miR-122 via toehold-promoted strand displacement reaction and enzyme-assisted cycle amplification

It is of great significance to develop a sensitive detection platform for miRNA, a non-invasive biological indicator for the diagnosis, treatment and prognosis monitoring of tumors. In this paper, we design an electrochemical DNA sensor to detect miR-122, based on toehold promoted strand displacement reaction, combined with enzyme assisted cycle amplification. In this strategy, when miR-122 and Exonuclease III (Exo III) coexist in the detection system, the toehold promotes the chain replacement reaction, and the miR-122 hybridizes with a stem-loop DNA, HC-DNA. Simultaneously, Exo III hydrolyzes part of HC-DNA and releases miR-122 and sign-DNA. Then, after multiple enzymatic cyclic amplification reactions (N Cycle), a large amount of Sign-DNA was released. Methylene blue (MB), as the signal molecule, is attached to sign-DNA, close to the surface of the electrode, and resulting in a increased electrochemical signal. Using this method, we can qualitatively analyze the concentration of miR-122 by the amplified electrochemical signals. The detection limit of miR-122 is 0.304 pM, lower than that of the many common large-scale instrumental analysis. Further, the method has good selectivity and anti-interference performance in complex environments. This electrochemical DNA biosensor has potential applications in highly sensitive detection of target molecules even concluding viruses.

Improvement in long-term stability of field effect transistor biosensor in aqueous environments using a combination of silane and reduced graphene oxide coating

A field-effect transistor (FET) biosensor that can directly detect target molecules is a promising diagnostic tool for healthcare and medical applications. However, the immersion of FET sensors in aqueous physiological environments for the long-term stability might damage the insulating layer of silicon dioxide, resulting in the degradation of the sensor response reproducibility. In this study, to improve the durability of the sensor response for long-term immersion in aqueous solution, we examined the effectiveness of reduced graphene oxide (rGO) coating on the gate insulator of FET sensors. The rGO coating was applied to the gate surface of the FET biosensor modified with an amino-terminated self-assembled monolayer by two steps: deposition of graphene oxide (GO) dispersion onto the monolayer-modified surface and subsequent thermal reduction of GO. The transconductance of both GO-coated and rGO-coated FETs remained unchanged after incubation under physiological conditions, suggesting that graphene prevents cations in the electrolytes from invading the gate insulator of the FET. Furthermore, functionalizing the rGO-coated FET surface enabled specific detection of target molecule.

A Simple Two-Dimensional Convective-Diffusive Based Modelling Study of a Biosensor for Glucose Detection

Over the past decade, novel technologies for biomolecular detection of target molecules of interest have been reported. The chemical reactions that govern the kinetics of the transport of target molecules to the sensor are also of significant importance. Here we analyze how the diffusion, convection, and reaction kinetics can drive the experimental design of a simple glucose biosensor. In this work, a physically intuitive and practical understanding of analyte transport is presented by modelling and simulating a two-dimensional convective-diffusive transport phenomenon in COMSOL Multiphysics to understand the mechanism of surface capture dynamics of a biosensor under the assumption of perfect binding kinetics. The target molecule of interest is glucose. In the model, 1 mol/m3 of the glucose solution is introduced at the inlet at 1 mm/s, and the process is simulated as a stationary study. It was found that by decreasing the flow rate, a threshold exists where the detection of target molecules by the sensor is solely controlled by diffusion. Comparison of convective and diffusive fluxes of the system as well as the concentration distribution reveal that a decrease in flow rate increases diffusive transport, thus increasing the fraction of target molecules that are captured by the sensor.

Electrochemical Signal Amplification Strategies and Their Use in Olfactory and Taste Evaluation.

Biosensors are powerful analytical tools used to identify and detect target molecules. Electrochemical biosensors, which combine biosensing with electrochemical analysis techniques, are efficient analytical instruments that translate concentration signals into electrical signals, enabling the quantitative and qualitative analysis of target molecules. Electrochemical biosensors have been widely used in various fields of detection and analysis due to their high sensitivity, superior selectivity, quick reaction time, and inexpensive cost. However, the signal changes caused by interactions between a biological probe and a target molecule are very weak and difficult to capture directly by using detection instruments. Therefore, various signal amplification strategies have been proposed and developed to increase the accuracy and sensitivity of detection systems. This review serves as a reference for biosensor and detector research, as it introduces the research progress of electrochemical signal amplification strategies in olfactory and taste evaluation. It also discusses the latest signal amplification strategies currently being employed in electrochemical biosensors for nanomaterial development, enzyme labeling, and nucleic acid amplification techniques, and highlights the most recent work in using cell tissues as biosensitive elements.

Detection of Atrazine and its metabolites by photonic molecularly imprinted polymers in aqueous solutions

Water contamination caused by atrazine (ATZ) application in agriculture is a risk to the environment and human health. Common analytical methods are expensive and complex. A sensor for low-cost and simple detection of ATZ and its metabolites, deethylatrazine (DEA) and deisopropylatrazine (DIA), in aqueous solutions was developed by combining colloidal crystal with molecular imprinting technique. The sensor is formed by 3D interconnected macroporous structure with numerous nanocavities derived from ATZ and its metabolites imprinting in a thin polymeric film. Molecularly Imprinted Polymers (MIPs) were characterized by Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) and were incubated in solutions at variable concentrations. Target molecules were specifically absorbed in nanocavities and caused swelling in the polymer resulting in changes of Bragg diffraction peak wavelength. Kinetic tests showed that rebinding equilibrium was reached within 20 minutes. The sensor had a dynamic range of 0.1 to 10 ppb for quantifying target analytes in aqueous solutions with limit of detection of 0.1, 0.2, and 0.3 ppb, and limit of quantification of 0.33, 0.66, and 1 ppb for ATZ, DEA, and DIA, respectively. Cross-reactivity tests were conducted in 1 and 5 ppb solutions combining all three targets and showed absence of positive interference effects and low probability of false positives given by individual sensors. MIPs were examined in natural waters containing ATZ and its metabolites contamination, showing good agreement with real concentrations of targets. The MIP yields rapid and efficient detection of target molecules in aqueous solutions close to environmentally relevant concentrations.

Quasi‐3D‐Structured Surface‐Enhanced Raman Scattering Substrates Based on Silver Nanoparticles/Mesoporous Silicon Hybrid

The surface‐enhanced Raman scattering (SERS) is a powerful spectroscopic technique for ultrasensitive and selective biochemical detection due to its capability of providing the “fingerprint” of information of molecular structures at low concentrations. Herein, mesoporous silicon (MPSi)‐based nanocomposites containing silver nanoparticles (AgNPs) provide novel efficient substrates for the detection of molecules. The quasi‐3D‐structured SERS substrate is formed by introducing Ag into the MPSi hollow column‐shaped nanostructure. This SERS substrate uniquely combines the ability of metal surfaces to amplify Raman scattering signals with an enlarged surface area and the increasing light–matter interaction duration that generates large SERS signals for the detection of chemicals. The proposed SERS substrate is verified by detecting orange methylene in solutions at ultralow concentration in the range of 10−11–10−6 m with a linear‐dependent coefficient of R2 = 0.99922 and the SERS enhancement factor is achieved up to 1010. Moreover, the proposed platform can detect diphenylamine with ultralow concentration of 10−10 m and reliable reproducibility with an average relative stand deviation of 6.3%. The ability for MPSi substrate to detect target molecules at low concentrations helps pave a way to develop the detection tool for integrated, on‐chip devices.

Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry

Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance.

Flexible Hydrophobic CFP@PDA@AuNPs Stripes for Highly Sensitive SERS Detection of Methylene Blue Residue.

Considering the inherent hydrophilic and porous nature of paper, the rapid absorption and diffusion of aqueous analyte solutions on paper-based SERS substrates may severely affect the Raman detection sensitivity and accuracy in the detection of target molecules. In this work, a series of hydrophobic CFP@PDA@AuNPs stripes were obtained through in situ synthesizing of gold nanoparticles (AuNPs) on a polydopamine (PDA)-decorated cellulose filter paper (CFP) and functionalized with perfluorodecanethiol (PFDT). When the SERS performance of the substrates was examined using 4-ATP, the hydrophobic CFP@PDA@AuNPs substrate showed superior sensitivity, reproducibility and stability due to the hydrophobic enrichment effect, with the detection limit decreasing to 10−9 M and the enhancement factor as high as 2.55 × 107. More importantly, it was feasible to apply the hydrophobic paper substrate as an excellent SERS sensor to detect methylene blue (MB) residues in lake water in a highly sensitive manner. The lowest detectable limit of MB was 100 nM, and it showed a low relatively standard deviation (RSD) value of 5.28%. Hydrophobic CFP@PDA@AuNPs stripes may serve as excellent sensors for target molecule detection and have tremendous potential in food security, and environmental and chemical detection.

Dynamic Single-Molecule Sensors: A Theoretical Study.

One of the key advantages of single-molecule sensors over conventional ensemble technologies is their capability of revealing the heterogeneity among molecular events. In dynamic single-molecule sensing, heterogeneity in molecular interaction kinetics is quantified as the fingerprint to specifically detect target molecules. This strategy offers a unique approach to develop ultrasensitive biosensors with a limit of detection at the fM level, which is three orders of magnitude lower than that of conventional assays. However, due to the lack of a comprehensive theoretical model, the rational design of dynamic single-molecule sensors is challenging. Herein, we present the theoretical study of sensing performance with a hydrodynamic model. We quantitatively show that there is a dilemma regarding the probe design. High-affinity probes offer higher specificity but require extremely long assay time, while low-affinity probes could shorten the assay time but are prone to the interference from unwanted molecules. This study also suggests that one possible solution to solve this dilemma is by applying external disturbance to the system, as we have recently demonstrated by experiments. We anticipate that this work could inspire the rational design of single-molecule sensors to further improve the sensitivity, specificity, and multiplexing capability.

CRISPR-Based Diagnostics for Point-of-Care Viral Detection

Point-of-care detection of viral infection is required for effective contact-tracing, epidemiological surveillance, and linkage to care. Traditional diagnostic platforms relying on either antigen detection or nucleic amplification are limited by sensitivity and the need for costly laboratory infrastructure, respectively. Recently, CRISPR-based diagnostics have emerged as an alternative, combining equipment light workflows with high specificity and sensitivity. However, as a nascent technology, several outstanding challenges to widespread field deployment remain. These include the need for pre-detection amplification of target molecules, the lack of standardization in sample preparation and reagent composition, and only equivocal assessments of the unit-economics relative to traditional antigen or polymerase chain reaction-based diagnostics. This review summarizes recent advances with the potential to overcome existing translational barriers, describes the events in CRISPR-based detection of target molecules, and offers perspective on how multiple approaches can be combined to decrease the limit of detection without introducing pre-amplification.

Recent Progress in Metal-Organic Framework Based Fluorescent Sensors for Hazardous Materials Detection.

Population growth and industrial development have exacerbated environmental pollution of both land and aquatic environments with toxic and harmful materials. Luminescence-based chemical sensors crafted for specific hazardous substances operate on host-guest interactions, leading to the detection of target molecules down to the nanomolar range. Particularly, the luminescence-based sensors constructed on the basis of metal-organic frameworks (MOFs) are of increasing interest, as they can not only compensate for the shortcomings of traditional detection techniques, but also can provide more sensitive detection for analytes. Recent years have seen MOFs-based fluorescent sensors show outstanding advantages in the field of hazardous substance identification and detection. Here, we critically discuss the application of MOFs for the detection of a broad scope of hazardous substances, including hazardous gases, heavy metal ions, radioactive ions, antibiotics, pesticides, nitro-explosives, and some harmful solvents as well as luminous and sensing mechanisms of MOF-based fluorescent sensors. The outlook and several crucial issues of this area are also discussed, with the expectation that it may help arouse widespread attention on exploring fluorescent MOFs (LMOFs) in potential sensing applications.

Acoustofluidics-manipulated triple-emission fluorescent nanoprobe aggregates with multicolor-variation for colorimetric quantitative assay

Acoustofluidics-assisted colorimetric method, a promising point-of-care test (POCT) strategy, has been applied in convenient detection of target molecules using the sensing nanomaterials, but remains a formidable challenge to design fluorescent probe with profuse color evolution, realizing sensitive, simple and accurate determination of analyte. Here, a novel triple-emission fluorescent probe is constructed through a simple blend of two ratiometric fluorescent probes (blue-organosilane-polymerized carbon dots@SiO2 nanoparticles@red-CdTe/CdS quantum dots (b@SiO2@r) and blue-organosilane-polymerized carbon dots@SiO2 nanoparticles@green-CdTe/CdS quantum dots (b@SiO2@g)) at the optimized volume ratio of 11:8. As signal reporters of the triple-emission probe, outer modified “r” and “g” will be quenched in sequence by target via two-step response strategy while the inner “b” as internal standard remains constant. When acoustic field is applied, the effect of acoustofluidics-based nanoparticles concentration makes triple-emission probe aggregate completely, accompanying fluorescence color signal being enhanced, sensitivity of colorimetric assay being improved and multicolor-variation (from orange to dark-goldenrod to dark-olive-green to sea-green to dark-cyan to final steel-blue) with the increase of analyte amount. For POCT demonstration, hemoglobin (Hb) is used as a model target and the captured fluorescence photos of triple-emission probe aggregates are analyzed by a chromaticity analysis application on the smartphone to calculate the red, green and blue values, which are used for accurate Hb quantification with the limit of detection of 1.99 mg/L. Whole test is finished within ∼12 min. The acoustofluidics-manipulated triple-emission probe biosensing strategy has also been applied to the rapid and visual quantitative detection of Hb in human blood.

In Situ Flow Cytometer Calibration and Single-Molecule Resolution via Quantum Measurement

Fluorescent biomarkers are used to detect target molecules within inhomogeneous populations of cells. When these biomarkers are found in trace amounts it becomes extremely challenging to detect their presence in a flow cytometer. Here, we present a framework to draw a detection baseline for single emitters and enable absolute calibration of a flow cytometer based on quantum measurements. We used single-photon detection and found the second-order autocorrelation function of fluorescent light. We computed the success of rare-event detection for different signal-to-noise ratios (SNR). We showed high-accuracy identification of the events with occurrence rates below even at modest SNR levels, enabling early disease diagnostics and post-disease monitoring.

Specific iodide effect on surface-enhanced Raman scattering for ultra-sensitive detection of organic contaminants in water

Ultra-sensitive detection of target molecules by surface-enhanced Raman scattering (SERS) is crucial in a wide range of fields but remains a great challenge. In this work, we report a simple and effective protocol for obtaining highly SERS-sensitive probe by mixing iodide with silver sol. The specific iodide effect on the SERS sensitivity is systematically investigated. It is found that, iodide can effectively promote the SERS enhancement of anionic and cationic analytes, and I- ion has a higher activating effect on SERS than that of Cl- ion. The as-prepared SERS-active substrate demonstrates excellent enhancement for rhodamine 6G with a high Raman enhancement factor of 1.8 × 108, which allows the detection limit of 1.0 × 10-13 M. Our findings in this work should be important for the developing of SERS theory and ultra-sensitive detection applications.