Presence Of Target Molecules Research Articles

A magnetic control enrichment technique combined with terahertz metamaterial biosensor for detecting SARS-CoV-2 spike protein

The highly contagious SARS-CoV-2 virus, responsible for the COVID-19 pandemic continues to pose significant challenges to public health. Developing new methods for early detection and diagnosis is crucial in combatting the disease, mitigating its impact and be prepared for future challenges in pandemic diseases. In this study, we propose a terahertz (THz) biosensing technology that capitalizes on the properties of THz metamaterial in conjunction with magnetic nanoparticles. This approach can accurately identify the SARS-CoV-2 spike protein by pinpointing its location on the THz resonance sources grooved surface. The magnetic nanoparticles are employed to selectively bind with target molecules, and migrate towards the THz metamaterial unit cell when exposed to an applied magnetic field. The presence of target molecules in to the metamaterial variation in the frequency, amplitude, and phase of the resonance response, thus enabling swift, accurate and sensitive detection. To assess the effectiveness of the proposed technique, we have conducted a comparative analysis between real samples on platforms controlled by magnetic manipulation and those without the control. It was confirmed that the proposed THz sensing method demonstrated a linear detection range spanning from 0.005 ng mL−1 to 1000 ng mL−1 with a detection limit of 0.002 ng mL−1. Furthermore, it exhibited a frequency shift of 24 GHz and a stability index of 95%. The THz biosensing technique may pave a new avenue in identifying and preempting the spread of potential pandemic diseases.

A pendulum-type electrochemical aptamer-based sensor for continuous, real-time and stable detection of proteins

Continuous detection of proteins is crucial for health management and biomedical research. Electrochemical aptamer-based (E-AB) sensor that relies on binding affinity between a recognition oligonucleotide and its specific target is a versatile platform to fulfill this purpose. Yet, the vast majority of E-AB sensors are characterized by voltammetric methods, which suffer from signal drifts and low-frequency data acquisition during continuous operations. To overcome these limitations, we developed a novel E-AB sensor empowered by Gold nanoparticle-DNA Pendulum (GDP). Using chronoamperometric interrogation, the developed sensor enabled drift-resistant, high-frequency, and real-time monitoring of vascular endothelial growth factor (VEGF), a vital signaling protein that regulates angiogenesis, endothelial cell proliferation and vasculogenesis. We assembled VEGF aptamer-anchored GDP probes to a reduced graphene modified electrode, where a fast chronoamperometric current transient occurs as the GDP rapidly transport to the electrode surface. In the presence of target molecules, longer and concentration-dependent time decays were observed because of slower motion of the GDP in its bound state. After optimizing several decisive parameters, including composition ratios of GDP, probe density, and incubation time, the GDP empowered E-AB sensor achieves continuous, selective, and reversible monitoring of VEGF in both phosphate buffered saline (PBS) solutions and artificial urine with a wide detection range from 13 fM to 130 nM. Moreover, the developed sensor acquires signals on a millisecond timescale, and remains resistant to signal degradation during operation. This study offers a new approach to designing E-AB architectures for continuous biomolecular monitoring.

Encoded Structural Color Microneedle Patches for Multiple Screening of Wound Small Molecules.

Detection of biomarkers associated with wound conditions provides in-depth healthcare information and benefits wound healing treatment. The current aim of wound detection is to achieve in situ multiple detection. Here, we describe novel encoded structural color microneedle patches (EMNs) combining photonic crystals (PhCs) and microneedle arrays (MNs) for multiple wound biomarker detection in situ. Using a partitioned and layered casting strategy, the EMNs can be divided into different modules and each serves for the detection of small molecules associated with chronic hard-to-healing wounds, including pH, glucose and histamine. pH sensing is based on the interaction between hydrogen ions and carboxyl groups from hydrolyzed polyacrylamide (PAM); glucose sensing is achieved with the help of glucose-responsive fluorophenylboronic acid (FPBA); while histamine sensing relies on specific recognition of aptamers and target molecules. Owing to the responsive volume change of these three modules in the presence of target molecules, the EMNs can create structural color change and characteristic peak shift of the PhCs, thus realizing the qualitative measurement of target molecules with a spectrum analyzer. It is further demonstrated that the EMNs behave well in the multivariate detection of rat wound molecules. These features indicate that the EMNs can be valuable smart detection systems for wound status screening. This article is protected by copyright. All rights reserved.

Abstract P5-02-21: RAB5 is a generic biomarker for ADC efficacy

Abstract Antibody-drug conjugates (ADCs) have demonstrated impressive activity in recent clinical trials in breast cancer. Such targeted therapeutics strongly depends on the presence of target molecules on the tumor cells, and the presence of such target molecules may determine the response of ADCs. However, ADCs are also dependent on cellular uptake, and factors regulating endocytosis as well as intracellular trafficking may strongly influence ADC activity. We have recently demonstrated that the activity of the HER2 targeted ADC trastuzumab emtansine (T-DM1) is dependent on the expression of RAB5A, a protein regulating endocytosis (1). A significant correlation between Rab5A expression and T-DM1 efficacy was found in a panel of HER2 expressing breast- and ovarian cancer cell lines. This result was verified in the I-SPY2 clinical trial where patients with high RAB5A expression were more likely to achieve a pathological complete response following T-DM1 as a neoadjuvant. The result was further validated in patients treated with T-DM1 in the Kamilla study where patients with a high RAB5A had a longer progression free survival. All ADCs should in principle be dependent on endocytosis to exert their activity. This triggered the investigation of proteins regulating endocytosis as predictive biomarkers for ADCs in general. METHODS: HER2-positive breast and ovarian cancer cell lines were evaluated with respect to the sensitivity and efficacy of treatment with T-DM1, trastuzumab deruxtecan, sacituzumab govitecan and the targeted toxin MH3B1/rGel.. The expression levels of proteins involved in endocytosis and endocytic trafficking including RAB4A, RAB5A and RAB11A were investigated in addition to the molecular drug targets (HER2 and TROP2). Cellular drug sensitivity was correlated to the expression levels of the investigated proteins using both RNA and protein as readout. RESULTS: The early endosome marker RAB5A, was found to correlate positively to the activity of trastuzumab deruxtecan, sacituzumab govitecan and MH3B1/rGel in the HER2 positive cell line panel, confirming the importance of RAB5A expression for the activity of these drugs. A significant correlation was found between RAB5A and drug efficacy using both protein and RNA as a readout. CONCLUSION: The present results indicate RAB5A as a generic predictive biomarker for both ADCs and targeted toxins which both depend on cellular uptake for cytotoxic efficacy. The results supports using both protein and RNA as a readout for RAB5A expression and point towards the development of a RAB5A stratification procedure for ADC and targeted toxin treatment. 1. Engebraaten O, Yau C, Berg K, Borgen E, Garred O, Berstad MEB, Fremstedal ASV, DeMichele A, Veer LV, Esserman L, Weyergang A. RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer. Nature communications 2021;12(1):6427 doi 10.1038/s41467-021-26018-z. Citation Format: Olav Engebraaten, Astrid Medhus, Ane Longva, Anette Weyergang, Kristian Berg. RAB5 is a generic biomarker for ADC efficacy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-21.

In situ electrochemical surface modification of Au electrodes for simultaneous label-free SERS detection of ascorbic acid, dopamine and uric acid

Simultaneous detection of biomolecules with high sensitivity and selectivity is highly demanded for point-of-care diagnosis. Here, a simple solution-based surface-enhanced Raman spectroscopy (SERS) method is presented to facilitate rapid and direct ternary detections of ascorbic acid (AA), dopamine (DA) and uric acid (UA) in urine. The approach focuses on in situ surface modification of Au nanopillar electrode by Au electrodeposition in the presence of target molecules. Formation of new plasmonic nanostructures on the nanopillar array effectively perturbs the biomolecules adsorbed on the Au surface and confines the target molecules in solution into active regions of the generated hotspots, resulting in the significant amplifications of Raman signals within 60 s. The method enabled reliable label-free detections of AA, DA, and UA at relevant physiological ranges with detection limits of 1, 0.1 and 1 nM, respectively. To the best of our knowledge, this is the first demonstration of simultaneous detection and quantification of the biomolecules by SERS. The practical applicability of this novel method was ascertained by the analysis with non-pretreated human urine.

Label‐Free Colorimetric Biosensors Based on Aptamers and Gold Nanoparticles: A Critical Review

AbstractTaking advantage of the adsorption of single‐stranded DNA oligonucleotides by gold nanoparticles (AuNPs) and the protection effect of the adsorbed DNA against salt‐induced aggregation of AuNPs, a label‐free colorimetric sensor for the detection of DNA was reported in 2004. Since then, the range of target molecules has extended from complementary nucleic acids to metal ions and small molecules by using aptamers. In the presence of target molecules, a blue color arising from aggregated AuNPs is expected. However, these sensors only considered aptamer binding to its target, and the adsorption of aptamers by AuNPs, while the target/AuNP interactions were ignored. We recently found that target adsorption can often dominate the system. In this Review, we list literature examples of using this label‐free strategy for sensing aptamer targets. Seven target analytes are discussed in detail. For As(III), dopamine, melamine, kanamycin, adenosine, and ATP, target adsorption dominated, and the same color change was observed even with non‐aptamer sequences. Only in the case of K+ detection, did the effect of specific aptamer binding dominate, attributable to weak K+/AuNP interactions. These examples call for a careful evaluation of target adsorption and the use of non‐aptamer control sequences in validating these sensors.

Simulations of Benzene and Hydrogen-Sulfide Gas Detector Based on Single-Walled Carbon Nanotube over Intrinsic 4H-SiC Substrate.

Carbon nanotubes (CNTs)-based sensors have gained significant importance due to their tremendous electrical and physical attributes. CNT-based gas sensors have high sensitivity, stability, and fast response time compared to that of solid-state sensors. On exposure to a large variety of organic and inorganic compounds, the conductivity of CNT changes. This change in electrical conductivity is being used as a detection signal to detect different target molecules. Hydrogen-sulfide and benzene are hazardous gases that can cause serious health issues in humans. Therefore, it is mandatory to detect their presence in industrial and household environments. In this article, we simulated CNT-based benzene and hydrogen-sulfide sensor with a nanoscale semiconductor device simulator—Quantumwise Atomistix Toolkit (ATK). The change in the device density of states, electric current, and photocurrent in the presence of target molecules have been calculated. The change in photocurrent in the presence of target molecules has been proposed as a novel detection mechanism to improve the sensor selectivity and accuracy. This change in photocurrent as well as electric current in the presence of target molecules can be used simultaneously as detection signals. Our intension in the future is to physically fabricate this simulated device and use photocurrent as well as electric current as detection mechanisms.

Core-satellite assemblies and exonuclease assisted double amplification strategy for ultrasensitive SERS detection of biotoxin

In this work, core-satellite assemblies and exonuclease assisted double amplification strategy is developed to produce surface-enhanced Raman scattering (SERS) biosensor towards ultrasensitive detection of biotoxin. In the presence of target molecules, the exonuclease III (Exo III) assisted efficient recycling amplification provides an excellent pathway for the fabrication of core-satellite SERS sensor. Briefly, the proposed strategy includes the following double amplifications: (i) Exo III induced target-related signal amplification; (ii) core-satellite assemblies assisted formation of SERS “hot-spots” induced signal amplification. To show the applicability of the suggested strategy, the detection of ochratoxin A (OTA), one of the most toxic and widely distributed biotoxin, is demonstrated as an example. The results show that the limit of detection (LOD) of OTA is 0.83 fg mL−1 (S/N = 3). On the basis of the DNA aptamer induced specific target recognition, hence our sensing strategy is easy to be expended to the ultrasensitive detection of other targets, e.g., DNAs, RNAs, and other molecules that have corresponding DNA aptamers.

Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid

Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

MoS2/nitrogen doped graphene hydrogels p-n heterojunction: Efficient charge transfer property for highly sensitive and selective photoelectrochemical analysis of chloramphenicol

Constructing junctions between semiconductors is an effective way to promote charge separation and thus to improve the photoelectrochemical (PEC) performances, and specifically, p-n heterojunction is considered as a very promising structure. Herein, we designed and fabricated MoS2/nitrogen doped graphene hydrogels (MoS2/NGH) p-n heterojunction by a facile one-pot hydrothermal route. The as-fabricaterd MoS2/NGH heterostructures demonstrated the excellent PEC activity, exhibiting enhanced photocurrent intensity by the fast transfer and separation rate of photogenerated electron-hole owing to the construction of p-n heterojunction. Based on the high PEC performances of the MoS2/NGH heterostructure, a novel sensitive PEC sensor was developed for the determination of chloramphenicol (CAP) with the assistance of aptamer. In the presence of target molecules, the as-fabricated PEC sensor could recognize the CAP quickly and then consume the holes in the interface of heterostructures, inhibiting the recombination of photogenerated electron-hole pairs, resulting the enhanced photocurrent. Specially, with the concentration of CAP increased, the photocurrent enhanced gradually. Excellent linearity was obtained in the concentration range from 32.3 ng/L to 96.9 μg/L, and the limit of detection was 3.23 ng/L. Moreover, the as-fabricated PEC sensor exhibited rapid response, high stability, low-cost and high selectivity, which could be successfully applied to the analysis of CAP in honeycomb samples.

Label-free aptasensor for ochratoxin A detection using SYBR Gold as a probe

A label-free sensing strategy employing aptamers, SYBR Gold, and exonuclease I (Exo I) for ochratoxin A (OTA) detection was designed. In the presence of target molecules (OTA), the conformation of the aptamer specific for OTA is switched from a random coil to an antiparallel G-quadruplex. Subsequently, Exo I is added into the mixture to digest the unfolded aptamers selectively, which are the preferred substrates of Exo I. Following the addition of SYBR Gold as probe, a strong fluorescence intensity is obtained. This aptasensor shows high selectivity toward OTA with a low limit of detection (16.5nM). The validity of the procedure and applicability of the aptasensor are successfully assessed through the detection of OTA in spiked red wine and beer without interference from the sample matrix. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples indicates its usefulness as a tool for verifying the effectiveness of mycotoxin control strategies.

Fluorescent Biosensors Based on Single-Molecule Counting.

Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescence-based single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the single-molecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery.

An aptamer based method for small molecules detection through monitoring salt-induced AuNPs aggregation and surface plasmon resonance (SPR) detection

A simple, rapid and sensitive method for small molecules detection based on surface plasmon resonance (SPR) is presented. In this work, aptamer and unmodified gold nanoparticles (AuNPs) were combined to construct a sensor for small molecules detection. The AuNPs/ssDNA complexes display excellent stability under high salt condition. However, in the presence of target molecules, the binding of aptamers/AuNPs would be broken, thus resulting in the aggregation of AuNPs due to the loss of protection by the aptamers. The aggregation of the unmodified AuNPs led to a change of refractive index of the solution. Thus, assay results can be obtained by a fiber optic surface plasmon resonance (FOSPR) probe. In order to improve the detection sensitivity, U-shaped fiber optic probe silver film coating was employed. This new method shows excellent analytical performance for bisphenol A (BPA) in the range of 0.01–50ng/mL (ppb) with a limit of detection (LOD) of 0.0037ng/mL (ppb), which is about 30 times better than that of conventional enzyme-linked immunosorbent assay (ELISA). In addition, the proposed method exhibited excellent specificity, acceptable precision, and good accuracy. The proposed SPR assay was successfully applied to BPA determination in actual samples. Therefore, the present method was demonstrated to be promising for effective detection of small molecules.

Evanescent Field Biosensor Using Polymer Slab Waveguide-Based Cartridges for the Optical Detection of Nanoparticles

We present a polymer optical waveguide integration technology for the detection of nanoparticles in an evanescent field (EF)-based biosensor. Polymer waveguides together with their light coupling structures are designed to be integrated in a novel cartridge concept which will eventually lead to cost-effective, rapid, and easy-to-use point-of-care testing (POCT). The selected slab waveguides generate a homogeneous and well-defined EF, illuminating magnetic nanoparticles that are used as optical contrast labels and are measured using dark-field microscopy. The nanoparticles quantitatively bind to the sensor surface in the presence of target molecules, mediated by antibodies. Compatibility of the waveguide materials with these biomolecules is therefore a strong requirement. When designing a biosensor for POCT, it is also necessary to consider fabrication and manipulation tolerances, targeting the compatibility with mass-production technologies. In this context, polymer optics offer unique advantages, and in combination with the optical contrast labels a very high sensitivity can be achieved. The sensing concept was assessed by comparing various commercially available polymer materials (LightLink, Ormocer, Epocore/Epoclad) in terms of waveguide design and fabrication, optical performance (detection of nanoparticles), and biological compatibility and performance as a sensor surface in an immuno-assay for cardiac troponin I.

Aptamer-based single-walled carbon nanohorn sensors for ochratoxin A detection

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most significant mycotoxins encountered in foods. In this research, a novel fluorescence biosensing strategy for simple and sensitive OTA detection using an aptamer specific for OTA as recognition element and single-walled carbon nanohorns (SWCNHs) as fluorescence quencher was reported. In the absence of target molecule (OTA), FAM (carboxyfluorescein)-modified aptamer was adsorbed onto the SWCNH surface. In this arrangement, the fluorophore and quencher were in close proximity; thus, FAM fluorescence was readily quenched by fluorescence resonance energy transfer from dye to carbon nanohorn. In the presence of target molecules (OTA), the OTA-specific aptamer form an antiparallel G-quadruplex, which is resistant to being wrapped onto SWCNHs. Therefore, the fluorescence of FAM cannot be quenched, and the fluorescent intensity as a function of OTA concentration was correspondingly measured. We obtained a detection limit of 17.2 nM for our sensing platform based on SWCNHs, with a linear detection range of 20–500 nM. The proposed assay system also exhibited high selectivity for OTA against other mycotoxins. The biosensor was verified for real sample application by testing 1% red wine containing buffer solution spiked with a series of OTA concentrations, and there is a good linear relationship (correlation coefficient = 0.990) between the fluorescence intensity and the logarithm of OTA concentration. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples indicated the sensor being a useful tool for verifying the effectiveness of mycotoxin control strategies.

One step forward, two steps back: The story of everolimus in advanced breast cancer

There has been a substantial surge of ‘targeted agents’ in contemporary anticancer drug armamentarium and some of these agents have revolutionized the outcome of cancer patients. However, on contrary to the nomenclature, not all new targeted agents are selected based on presence of target molecules on the cancer cells. Drugs are typically approved based on demonstration of benefit in randomized controlled trials with regards to efficacy outcomes although both the ‘benefits’ and ‘outcomes’ are defined inconsistently. Surrogates that are not validated properly are often used as endpoints. Furthermore, new anticancer drugs are frequently associated with increased inconvenience to the patients and/or to the society due to added toxicity and cost. In this perspective article, emphasis is given to the above problems focusing on room for improvement in anticancer drug development. An illustration of a recently approved drug to treat advanced breast cancer, everolimus and a previously revoked drug bevacizumab is given.

Air-band optical resonators in one-dimensional Si photonic crystal waveguides for biosensing applications

Optical cavities using air-band states in Si photonic crystals (PhCs) are proposed as biosensors. Since the light in the air band of PhCs is preferably located at the lower-index cladding rather than the inside of higher-index Si, interactions are enhanced between the light and the target molecules in the cladding, being effective for the sensitive detections. Such an air-band PhC is used as an optical cavity, which is surrounded with another PhC as the photonic-bandgap mirrors. Simulations are performed for air-band resonators in one-dimensional (1D) PhC of arrayed holes in a Si channel waveguide, in order to analyze a figure of sensing, i.e., red shifts in the resonance wavelengths induced by the presence of target molecules. The S value, the amount of red shift caused by the unit change in the cladding index, can be as large as 200 nm/RIU, which is 2 times larger than those for ordinary 1D-PhC resonators. Fabricated devices successfully show the resonance peaks, whose wavelengths and quality factors agree well with theoretical ones. These results suggest that the air-band resonators are promising for high-performance biosensors.

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers

We have designed a single-stranded DNAzyme–aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3′ end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme–aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.

A label-free electrochemical biosensor for trace uranium based on DNAzymes and gold nanoparticles

A novel and highly sensitive electrochemical DNAzymes biosensor was fabricated using Au nanoparticles (AuNPs) immobilized on the surface of Au electrode that had been previously modified with self-assembled monolayers of 1,6-hexanedithiol. Different modified electrodes were prepared and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The AuNPs were found to have a large surface area to anchor a large number of negatively charged phosphate backbones of DNAzymes, which further absorbed the electroactive indicator of hexaammineruthenium(III) ([Ru(NH3)6]3+) to amplify the electrochemical signal. In the presence of target molecules, a large amount of DNA partly associated with [Ru(NH3)6]3+ were removed from the electrode surface, leading to a significant decrease in peak current. Differential pulse voltammetry signals of [Ru(NH3)6]3+ provided quantitative measures of the concentrations of uranyl ion (UO2 2+), with linear calibration ranging from 13 pM to 0.15 nM and a detection limit of 5 pM. The presence of other metal ions did not affect the detection of UO2 2+, which indicated the high specificity of UO2 2+. Therefore, a new electrochemical DNAzymes sensor was designed with specific DNAzymes and AuNPs as immobilization platform and signal amplifier.

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol.