Non-target Molecules Research Articles

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Novel lateral flow assay to detect H2O2 by utilizing self-biotinylation of G-quadruplex

We herein describe a novel lateral flow assay (LFA) to detect H2O2 by utilizing self-biotinylation of G-quadruplex (G4). In this strategy, the G4 strand promotes the self-biotinylation of G4 itself in the presence of H2O2, which is then allowed to bind to the FAM-labeled complementary detector probe. The resulting biotin-labeled G4/FAM-detector probe complex is captured on the test line, producing a red-colored band during lateral flow readout. Based on this unique approach, we achieved the naked-eye detection of target H2O2 at concentrations as low as 1 μM, with reliable quantification down to 0.388 μM. This method also demonstrated exceptional specificity in distinguishing H2O2 from other non-target molecules. We further verified its versatile applicability by reliably identifying another biomolecule, choline, by coupling with choline oxidase, which generates H₂O₂ during oxidation. This novel LFA strategy holds great promise as a powerful point-of-care testing (POCT) platform for detecting a large spectrum of target biomolecules by employing their corresponding oxidases.

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

In vitro selection of a trans aptamer complex for target-responsive fluorescence activation

BackgroundMost biological molecular complexes consist of multiple functional domains, yet rationally constructing such multifunctional complexes is challenging. Aptamers, the nucleic acid-based functional molecules, can perform multiple tasks including target recognition, conformational changes, and enzymatic activities, while being chemically synthesizable and tunable, and thus provide a basis for engineering enhanced functionalities through combination of multiple units. However, the conventional approach of simply combining aptamer units in a serial manner is susceptible to undesired crosstalk or interference between the aptamer units and to false interactions with non-target molecules; besides, the approach would require additional mechanisms to separate the units if they are desired to function independently. It is clearly a challenge to develop multi-aptamer complexes that preserve independent functions of each unit while avoiding undesired interference and non-specific interactions. ResultsBy directly in vitro selecting a ‘trans’ aptamer complex, we demonstrate that one aptamer unit (‘utility module’) can remain hidden or ‘inactive’ until a target analyte triggers the other unit (‘sensing module’) and separates the two aptamers. Since the operation of the utility module occurs free from the sensing module, unnecessary crosstalk between the two units can be avoided. Because the utility module is kept inactive until separated from the complex, non-specific interactions of the hidden module with noncognate targets can be naturally prevented. In our demonstration, the sensing module was selected to detect serotonin, a clinically important neurotransmitter, and the target-binding-induced structure-switching of the sensing module reveals and activates the utility module that turns on a fluorescence signal. The aptamer complex exhibited a moderately high affinity and an excellent specificity for serotonin with ∼16-fold discrimination against common neurotransmitter molecules, and displayed strong robustness to perturbations in the design, disallowing nonspecific reactions against various challenges. SignificanceThis work represents the first example of a trans aptamer complex that was in vitro selected de novo. The trans aptamer complex selected by our strategy does not require chemical modifications or immediate optimization processes to function, because the complex is directly selected to perform desired functions. This strategy should be applicable to a wide range of functional nucleic acid moieties, which will open up diverse applications in biosensing and molecular therapeutics.

A personal glucose meter-utilized strategy for portable and label-free detection of hydrogen peroxide

Rapid and precise detection of hydrogen peroxide (H2O2) holds great significance since it is linked to numerous physiological and inorganic catalytic processes. We herein developed a label-free and washing-free strategy to detect H2O2 by employing a hand-held personal glucose meter (PGM) as a signal readout device. By focusing on the fact that the reduced redox mediator ([Fe(CN)6]4-) itself is responsible for the final PGM signal, we developed a new PGM-based strategy to detect H2O2 by utilizing the target H2O2-mediated oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- in the presence of horseradish peroxidase (HRP) and monitoring the reduced PGM signal in response to the target amount. Based on this straightforward and facile design principle, H2O2 was successfully determined down to 3.63 μM with high specificity against various non-target molecules. We further demonstrated that this strategy could be expanded to identify another model target choline by detecting H2O2 produced through its oxidation promoted by choline oxidase. Moreover, we verified its practical applicability by reliably determining extracellular H2O2 released from the breast cancer cell line, MDA-MB-231. This work could evolve into versatile PGM-based platform technology to identify various non-glucose target molecules by employing their corresponding oxidase enzymes, greatly advancing the portable biosensing technologies.

Low-temperature iodinated plasmonically tuned Ag@AgI core-shell nanostructured thin films for highly specific ascorbic acid SERS detection at ultra-low concentrations

A simple low temperature iodination route was developed to controllably iodinate silver nanoislands film (Ag) into silver@silver iodide core-shell nanoparticles film (Ag@AgI) as a chemically stable, near infrared active surface enhanced Raman scattering (SERS) substrate. The localised surface plasmon resonance (LSPR) of the core-shell film exhibits a huge red-shift of 100 nm. The formation of Ag core and a complete ultra-thin AgI shell layer on top of the core is confirmed by transmission electron microscope. Selected area electron diffraction reveals the amorphous nature of AgI shell layer. These results are further substantiated by Raman and x-ray diffraction measurements. The enhancement factor of Ag@AgI film estimated with methylene blue is 5 × 106, an order of magnitude higher compared to that of Ag film. It demonstrated excellent chemical stability and unwavering SERS performance for two months since fabrication. The Ag@AgI film fielded as a label-free SERS substrate exhibited a lowest limit-of-detection of 1 × 10−12 M for ascorbic acid and a linear detection range of 1 × 10−6 to 1 × 10−12 M. The ascorbic acid selective signal enhancement of Ag@AgI film at low concentration region offers a practical sample dilution strategy to suppress interference from non-target molecules in biofluids.

Hydrogel-based Bioelectronic Tongue for the Evaluation of Umami Taste in Fermented Fish.

Bioelectronic tongues based on umami taste receptors have recently been reported for versatile applications such as food analyses. However, their practical applications are still limited, partly due to their limited stability and non-specific responses in real sample environments. Herein, we have developed a hydrogel-based bioelectronic tongue for the sensitive assessment of umami intensity in fish extract samples. In this study, the T1R1 venus flytrap of an umami taste receptor was immobilized on the gold floating electrodes of a carbon nanotube-based field-effect transistor. A polyacrylamide conducting hydrogel film was further hybridized on the sensor surface via physical adsorption, which could provide a good physiological environment to maintain the activity of receptors due to its excellent hydrophilicity and biocompatibility. The bioelectronic tongue with a receptor-embedded hydrogel structure showed a sensitive detection of umami substances down to 1 fM, and it also had a wide detection range of 10-15-10-2 M for monosodium glutamate and disodium inosinate, which covers the human taste threshold. More importantly, the proposed sensor could significantly reduce the non-specific binding of non-target molecules to a carbon nanotube channel as well as exhibit long-term stability, enabling sensitive detection of umami substances even in fish extract samples. Our hydrogel-based bioelectronic tongue provides a promising platform for future applications such as the flavor evaluation of foods and beverages.

Affinity-based electrochemical biosensor with antifouling properties for detection of lysophosphatidic acid, a promising early-stage ovarian cancer biomarker

Electrochemical techniques are considered to be highly sensitive, capable of fast response and can be easily miniaturized, properties which can aid with regard to the fabrication of compact point-of-care medical devices; however, the main challenge in developing such a tool is overcoming a ubiquitous, problematic phenomenon known as non-specific adsorption (NSA). NSA is due to the fouling of non-target molecules in the blood on the recognition surface of the device. To overcome NSA, we have developed an affinity-based electrochemical biosensor using medical-grade stainless steel electrodes and following a unique and novel strategy using silane-based interfacial chemistry to detect lysophosphatidic acid (LPA), a highly promising biomarker, which was found to be elevated in 90 % of stage I OC patients and gradually increases as the disease progresses to later stages. The biorecognition surface was developed using the affinity-based gelsolin-actin system, which was previously investigated by our group to detect LPA using fluorescence spectroscopy. We demonstrate the capability of this label-free biosensor to detect LPA in goat serum with a detection limit of 0.7 µM as a proof-of-concept for the early diagnosis of ovarian cancer.

Sensing Antibiotics in Wastewater Using Surface-Enhanced Raman Scattering.

Rapid and cost-effective detection of antibiotics in wastewater and through wastewater treatment processes is an important first step in developing effective strategies for their removal. Surface-enhanced Raman scattering (SERS) has the potential for label-free, real-time sensing of antibiotic contamination in the environment. This study reports the testing of two gold nanostructures as SERS substrates for the label-free detection of quinoline, a small-molecular-weight antibiotic that is commonly found in wastewater. The results showed that the self-assembled SERS substrate was able to quantify quinoline spiked in wastewater with a lower limit of detection (LoD) of 5.01 ppb. The SERStrate (commercially available SERS substrate with gold nanopillars) had a similar sensitivity for quinoline quantification in pure water (LoD of 1.15 ppb) but did not perform well for quinoline quantification in wastewater (LoD of 97.5 ppm) due to interferences from non-target molecules in the wastewater. Models constructed based on machine learning algorithms could improve the separation and identification of quinoline Raman spectra from those of interference molecules to some degree, but the selectivity of SERS intensification was more critical to achieve the identification and quantification of the target analyte. The results of this study are a proof-of-concept for SERS applications in label-free sensing of environmental contaminants. Further research is warranted to transform the concept into a practical technology for environmental monitoring.

Quantitative analysis of aqueous biomolecular mixtures by THz spectroscopy based on high-power LiNbO3 radiation source

The rapid and accurate identification of the types and contents of early pathological markers by THz technology are of particular importance for the prevention and treatment of major diseases. Nevertheless, these markers usually contain interference from water and other non-target molecules, resulting in low signal-to-noise ratio (SNR) and making identification and quantitative analysis difficult. Here, based on THz spectroscopy from a high-power THz source radiated by LiNbO3, we perform quantitative and real-time THz detection of mixtures (α-lactose monohydrate and 4-aminobenzoic acid) in liquids. The results demonstrate that the absorption spectra of the aqueous biomolecular mixtures exhibit an accumulation of THz features of each pure product, i.e., the amplitude of the absorption peaks is proportional to the mixing ratio, while the corresponding absorption baseline increases with decreasing concentration. Furthermore, the content of the target substance can be calculated from the linear relationship between the absorption spectra of pure and mixed samples. This technology will support the future application of THz-TDS in early disease diagnosis under complex states and environments.

Prussian blue analogues based polymer monolith with amphiphilic interface to construct highly selective and sensitive imidacloprid chemosensor

The most widely used assays for pesticides currently rely on the inhibition of natural enzymes, which are particularly sensitive to the surrounding environment, leading to some unreliable results. Up till now, there are few studies explored chemical detection methods for these stable pesticides. Here, we reported a novel chemosensor system and polymer materials for highly sensitive detection of imidacloprid pesticide. The polymer monolith with tunable surface hydrophilicity allows solvent-dependent adsorption of low-abundance target pesticide molecule. Prussian blue particles with Fenton catalytic activity were introduced into the polymeric monolith through Pickering emulsion, which capable of degrading the pesticide adsorbed on the column surface into easily detectable ions (NO3−, Cl−). This all-in-one functional material can minimize the interference of other non-target molecules through dual-channel detection, enabling sensitive detection of imidacloprid pesticide (30 ppb). We believe that the material described in this paper demonstrates for the first time the combination of an active polymer monolith for the enrichment and catalytic degradation with sensitive detection of pesticides, and this protocol can be used to develop a range of pesticides chemical sensing methods in the future.

Mass spectral profiling of caseinomacropeptide extracted from feeding material and jejunal fluid using three methods–ethanol precipitation, perchloric acid precipitation, and ultrafiltration

The ability of bovine κ-casein-derived caseinomacropeptide (CMP) to exert bioactivity in the human gut depends on its digestive survival. Sampling from the human jejunum after feeding CMP and top-down glycopeptidomics analysis facilitates the determination of CMP survival. To reduce interference from non-target molecules in mass spectrometric analysis, CMP must be isolated from digestive fluid. To identify an optimal extraction method, this study compared the profiles of CMP extracted from feeding material (commercial CMP in water) and digestive fluid by ethanol precipitation, perchloric acid (PCA) precipitation, and ultrafiltration. Ethanol precipitation yielded the highest ion abundances for aglycosylated CMP and glycosylated CMP in both feeding material and jejunal samples. Notably, PCA precipitation yielded the highest abundance of partially digested CMP-derived fragments in jejunal samples. Overall, ethanol precipitation was the most effective among the methods tested for intact CMP extraction from jejunal fluids, whereas PCA precipitation was optimal for extraction of CMP fragments.

Construction of PEGylated boronate-affinity-oriented imprinting magnetic nanoparticles for ultrasensitive detection of ellagic acid from beverages.

Molecularly imprinted polymers (MIPs) can exhibit antibody-level affinity for target molecules. However, the nonspecific adsorption of non-imprinted regions for non-target molecules limits the application range of MIPs. Herein, we fabricated PEGylated boronate-affinity-oriented ellagic acid-imprinting magnetic nanoparticles (PBEMN), which first integrated boronate-affinity-oriented surface imprinting and sequential PEGylation for small molecule-imprinted MIPs. The resultant PBEMN possess higher adsorption capacity and faster adsorption rate for template ellagic acid (EA) molecules than the non-PEGylated control. To prove the excellent performance, the PBEMN were linked with hydrophilic boronic acid-modified/fluorescein isothiocyanate-loaded graphene oxide (BFGO), because BFGO could selectively label cis-diol-containing substances by boronate-affinity and output ultrasensitive fluorescent signals. Based on a dual boronate-affinity synergy, the PBEMN first selectively captured EA molecules by boronate-affinity-oriented molecular imprinted recognition, and then the EA molecules were further labeled with BFGO through boronate-affinity. The PBEMN linked BFGO (PBPF) strategy provided ultrahigh sensitivity for EA molecules with a limit of detection of 39.1fgmL-1, resulting from the low nonspecific adsorption of PBEMN and the ultrasensitive fluorescence signal of BFGO. Lastly, the PBPF strategy was successfully employed in the determination of EA concentration in a spiked beverage sample with recovery and relative standard deviation in the range of 96.5 to 104.2% and 3.8 to 5.1%, respectively. This work demonstrates that the integration of boronate-affinity-oriented surface imprinting and sequential PEGylation may be a universal tool for improving the performance of MIPs.

Multifunctional membranes for lipidic nanovesicle capture

Tangential flow filtration membrane systems are employed for the isolation and concentration of extracellular vesicles. However, interfacial interactions between the membrane surface and species influence the flux and membrane performance. Here we propose a strategy aimed at introducing functional ligands over the membrane surface to improve the separation process through combined size-exclusion and affinity-based mechanisms, avoiding the binding of contaminants and other non-target molecules. Polysulfone membranes were modified by a nanometric coating of differently functionalized copolymers with the dual purpose of limiting non-specific interactions while promoting the chemoselective conjugation of a membrane-sensing peptide ligand (BPt) for lipid nanovesicles capture. Copoly azide polymer coating positively affects the physico-chemical properties of the membrane, improving filtration performance and antifouling capacity. A decrease of the flux decline ratio from 38.7 ± 3.9% to 21.2 ± 2.4% and an increase of the ratio of protein permeate concentration (Cp) to the respective feed concentration (Cf) to values of 0.97 was measured after coating the membrane with c-(DMA-N3-BP-MAPS) highlighting its capability to reduce protein adsorption. In addition, the BPt-functionalized membrane displayed a high capturing efficiency towards synthetic liposomes which, notably, can be promptly released upon mild treatment with a divalent cation solution. Overall, our work integrates conventional TFF principles with affinity-based isolation, broadening TFF perspective applications.

ABCB1 Can Actively Pump-out the Background-Free Tame Fluorescent Probe CO-1 From Live Cells.

Cell labelling using a small fluorescent probe is an important technique in biomedical sciences. We previously developed a biocompatible and membrane-permeable probe, CO-1, which has low nonspecific binding affinity towards nontarget molecules. Although this background-free tame probe has been utilized for labelling of various intracellular biomolecules in live cells, the probes' backgroung-free staining mechanism was not fully understood. Here, we propose that Gating-Oriented Live-cell Distinction (GOLD) mechanism occurs when ABCB1 transporter removes unbound CO-1 molecules from mammalian cells and, in a minor role, DIRC2 pumps CO-1 out from lysosomes. We also showed that solute carrier transporters were not involved in carrying CO-1 inside of cells. The role of reporters in assisting the probes' influx-efflux was analyzed by the combination of CRISPR library sceenings and inhibitors test. In summary, tame probe CO-1 cellular staining occurs in a dual mechanism where the probe moves freely through the cells membrane, but its washable property can be directly related to the action of ABCB1 transporter.

Disruption of IFNγ, PRF1, GZMB, or LYST results in reduced suppressive function in human CD8+ T cells

Abstract Human CD8+ T-cells play an important immune regulatory role during autoimmune diseases, characterized by suppressing the activation and proliferation of pathogenic effector cells, mediated in part through cytotoxicity (PRF/GZM), IFNγ secretion or by Fas/FasL interactions. Recent studies from our group demonstrated that IL-12 exposure during in vitro differentiation of naïve CD8+ T-cells induced an immune-suppressive deficit, with Tc1 cells showing significant loss of suppressive ability against CD4+ T-cells. RNAseq analysis revealed that various cytokines, transcription factors, and other regulatory molecules were differentially regulated in these cells. In this study, we further investigated the importance of IFNγ-, PRF1-, GZMB- and LYST-associated pathways in the suppressive ability of CD8+ T-cells. Using the CRISPR-Cas9 RNP optimized transfection system in primary bulk human CD8+ T-cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry when possible, as well confirmation of indels at genomic target sites. We observed that knockout of each of the four molecules, but not the knockout of IL-4 or IL-5, resulted in significantly diminished suppressive ability, as measured through in vitro immune suppression assays. Interestingly, in some of the knockouts, we observed differential expression of non-target molecules indicating potential functional links between these pathways. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T-cell-mediated suppression that could be targeted for immunotherapeutic intervention. This work was supported, in part, by grant awards to NJK from the NIH (R01 AI121567) and VA.

Target-induced recycling assembly of split aptamer fragments by DNA toehold-mediated displacement for the amplified colorimetric detection of estradiol

Estradiol is an endogenous hormone that affects many physiological functions in humans; thus, the demand for its detection has been increasing. Gold nanoparticle (AuNP)-based colorimetric sensors with split aptamers offer an easy-to-operate detection method for estradiol. However, the assay performance is still unmet. Here, we report a simple and sensitive colorimetric assay for the detection of estradiol, based on the target-induced recycling assembly of split aptamer fragments. This method contains three DNA fragments that remain in a metastable state without a target. After adding estradiol, the split aptamers assemble with the target, which can be regenerated by helper DNA, resulting in the formation of a three-way junction (3WJ)-like structure and the recycling of recognition. The generation of the 3WJ-like DNA structure causes the formation of unstable AuNPs, enhancing the salt-induced aggregation of AuNPs. The assay shows a detection limit of 0.7 nM, which is better than those of other aptamer-based detection methods. Moreover, the assay enables the detection of estradiol spiked in urine by avoiding any interference from contaminants. The colorimetric assay includes anti-estradiol DNA split aptamers, accounting for no false responses to non-target molecules. Therefore, the results demonstrated that it has application potential for the detection of estradiol in a complex matrix.

Inverse-designed waveguide-based biosensor for high-sensitivity, single-frequency detection of biomolecules.

Integrated silicon photonic waveguide biosensors have shown great potential for detecting bio-molecules because they enable efficient device functionalization via a well-developed surface chemistry, as well as simple scalable manufacturing, which makes them particularly suitable for low-cost point-of-care diagnostic. The on-chip integrated biosensors can be broadly classified into two types: (i) high-quality factor resonator sensors and (ii) interferometric sensors relying on non-resonant optical elements such as e.g. integrated waveguides. The former type usually requires a broadband or a tunable light source as well as complicated signal post-processing to measure a shift of the resonance frequency, while the latter exhibits a relatively low sensitivity due to the lack of efficient light recycling and phase accumulation mechanism in low quality factor elements. Additionally, high quality factor resonant photonic structures can be very sensitive to the presence of other non-target molecules in the water solution, causing sensor vulnerability to any noise. In this work, we combine a computational "inverse design" technique and a recently introduced high-contrast probe cleavage detection (HCCD) technique to design and optimize waveguide-based biosensors that demonstrate high sensitivity to the target molecule while being less sensitive to noise. The proposed biosensors only require a single frequency (or narrow-band) source and an intensity detector, which greatly simplifies the detection system, making it suitable for point-of-care applications. The optimal integrated sensor design that we demonstrate shows 98.3% transmission for the positive (target detected, probes cleaved) state and 4.9% transmission for the negative (probes are still attached) state at 1550nm wavelength. The signal intensity contrast (20.06-fold transmission increase) shown in this work is much greater than the shift of the resonance frequency (less than 1% wavelength shift) observed in conventional ring-resonator-based biosensors. The new design may pave the way for realizing a single-frequency highly sensitive and selective optical biosensor system with a small physical footprint and a simple optical readout on a silicon chip.

PEGylation of boronate-affinity-oriented surface imprinting magnetic nanoparticles with improved performance

High specific selectivity is the continuous goal of exploit glycoprotein-imprinted materials. Boronate-affinity-oriented surface imprinting can limit the heterogeneity of imprinted cavities, and PEGylation can reduce the nonspecific adsorption of imprinted materials towards non-target molecules. However, there are no reports on the integration of the above two advantages. Herein, we first integrated the boronate-affinity-oriented surface imprinting and PEGylation, and fabricated PEGylated boronate-affinity-oriented surface imprinting magnetic nanoparticles (PBSIMN) with horseradish peroxidase (HRP) as a model glycoprotein template. The successful synthesis of PBSIMN was demonstrated in detail by various characterization. Compared with non-PEGylated control, the PBSIMN showed greater adsorption capacity for HRP, and faster adsorption rate. To evaluate the improved performance, the PBSIMN was linked with hydrophilic boronic acid-modified/fluorescein isothiocyanate-loaded graphene oxide (BFGO), and used for the detection of HRP in real samples. Because PEGylation led to decrease of non-specific binding on PBSIMN, the proposed strategy provided ultrahigh sensitivity with limit of detection of 6.0 fg mL−1 for HRP, which were an order of magnitude lower than the non-PEGylated counterparts. When spiked with 0.05, 0.5 and 5.0 mg mL−1, recoveries of HRP were in the range of 97.4%–101.8% with relative standard deviation (RSD) no more than 5.4% for mouse serum, and between 98.2% and 103.2% with RSD no more than 5.0% human serum. This work indicates that the boronate-affinity-oriented surface imprinting and PEGylation can improve the performance of imprinted materials.

Simultaneous ultrasensitive detection of two breast cancer microRNA biomarkers by using a dual nanoparticle/nanosheet fluorescence resonance energy transfer sensor

The ability to detect multiple microRNAs (miRNAs) as cancer biomarkers with high sensitivity and good compatibility is still challenging. Here, we showed an ultrasensitive sensing system based on the fluorescence resonance energy transfer (FRET) effect between green-emitting and blue-emitting core@shell upconversion nanoparticles (UCNPs) and MoS2 nanosheets for simultaneously detecting two miRNAs. The core@shell UCNPs and MoS2 nanosheets served as energy donors and quenchers, respectively. The green-emitting and blue-emitting core@shell UCNPs were modified with single-stranded DNAs complementary to two breast cancer miRNA biomarkers, miR-593 and miR-155, respectively. When the two target miRNAs were absent in the samples, the ssDNA-modified nanoparticles bound to the MoS2 nanosheets directly through van der Waals forces, leading to the close proximity between the nanoparticles and nanosheets and thus the occurrence of FRET between them. When the two target miRNAs were present in the samples, each of the miRNAs hybridized with the complementary ssDNA on the nanoparticles, preventing the direct contact between the nanoparticles and the nanosheets and thereby causing miRNA concentration-dependent fluorescence recovery of the nanoparticles. Thus, monitoring the fluorescence recovery from the nanoparticles established a unique linear relationship between the fluorescence recovery and the target miRNA concentrations, allowing us to determine the concentration of the miRNAs by simply detecting the fluorescent signals. Our FRET sensor could reach an ultralow limit of detection (∼0.17 nM and ∼0.25 nM for miR-593 and miR-155, respectively). It could selectively detect the target miRNAs but not the non-target molecules. Our sensor can be extended to the ultrasensitive detection of other biomolecules and holds promise for the early diagnosis of diseases such as breast cancer.