Evaluating early apoptosis-related cellular MiRNAs with an ultrasensitive electrochemiluminescence nanoplatform.

It is known that the abnormal expression of specific cellular miRNAs is closely related to cell apoptosis, and so monitoring the level change of these miRNAs can in principle be used to evaluate the process of apoptosis stimulated by drugs. Towards this goal, here we construct an ultrasensitive electrochemiluminescence (ECL) nanoplatform via the target miRNA-triggered immobilization of spherical nucleic acid enzymes (SNAzymes) onto tetrahedral DNA nanostructures on the electrode surface, which catalyzes the luminol-H2O2 reaction to output an ECL signal. This enables the sensitive and specific detection of two apoptosis-related miRNAs, miR-21 and miR-133a, with a detection limit of 33 aM. Furthermore, we employed the developed ECL nanoplatform to monitor the levels of these two miRNAs inside cancer cells stimulated by DOX, showing that the level of miR-21 decreases, while that of miR-133a increases in the early apoptotic cells. This difference highlights the distinct roles of the two target miRNAs, where miR-21 promotes the early apoptosis of cancer cells, whereas miR-133a suppresses it, providing new insight into cell physiological processes.

Hairpin-like siRNA-Based Spherical Nucleic Acids.

The therapeutic use of small interfering RNAs (siRNAs) as gene regulation agents has been limited by their poor stability and delivery. Although arranging siRNAs into a spherical nucleic acid (SNA) architecture to form siRNA-SNAs increases their stability and uptake, prototypical siRNA-SNAs consist of a hybridized architecture that causes guide strand dissociation from passenger strands, which limits the delivery of active siRNA duplexes. In this study, a new SNA design that directly attaches both siRNA strands to the SNA core through a single hairpin-shaped molecule to prevent guide strand dissociation is introduced and investigated. This hairpin-like architecture increases the number of siRNA duplexes that can be loaded onto an SNA by 4-fold compared to the original hybridized siRNA-SNA architecture. As a result, the hairpin-like siRNA-SNAs exhibit a 6-fold longer half-life in serum and decreased cytotoxicity. In addition, the hairpin-like siRNA-SNA produces more durable gene knockdown than the hybridized siRNA-SNA. This study shows how the chemistry used to immobilize siRNA on nanoparticles can markedly enhance biological function, and it establishes the hairpin-like architecture as a next-generation SNA construct that will be useful in life science and medical research.

Label-Free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method-termed SpliMLiB for Split-and-Mix Library on Beads-was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.

Salt concentration modulates the DNA target search strategy of NdeI.

DNA target search is a key step in cellular transactions that access genomic information. How DNA binding proteins combine 3D diffusion, sliding and hopping into an overall search strategy remains poorly understood. Here we report the use of a single molecule DNA tethering method to characterize the target search kinetics of the type II restriction endonuclease NdeI. The measured search rate depends strongly on DNA length as well as salt concentration. Using roadblocks, we show that there are significant changes in the DNA sliding length over the salt concentrations in our study. To explain our results, we propose a model including cycles of 3D and 1D search in which salt concentration modulates the strategy by varying the length of DNA probed per 1D scan. At low salt NdeI makes a single non-specific encounter with DNA followed by an effective and complete 1D scan. At higher salt, NdeI must execute multiple cycles of target search due to the reduced efficacy of 1D search.

Fabrication of deferasirox-decorated aptamer-targeted superparamagnetic iron oxide nanoparticles (SPION) as a therapeutic and magnetic resonance imaging agent in cancer therapy.

In the current study, the synthesis of a theranostic platform composed of superparamagnetic iron oxide nanoparticles (SPION)-deferasirox conjugates targeted with AS1411 DNA aptamer was reported. In this regard, SPION was amine-functionalized by (3-aminopropyl)trimethoxysilane (ATPMS), and then deferasirox was covalently conjugated onto its surface. Finally, to provide guided drug delivery to cancerous tissue, AS1411 aptamer was conjugated to the complex of SPION-deferasirox. The cellular toxicity assay on CHO, C-26 and AGS cell lines verified higher cellular toxicity of targeted complex in comparison with non-targeted one. The evaluation of in vivo tumor growth inhibitory effect in C26 tumor-bearing mice illustrated that the aptamer-targeted complex significantly enhanced the therapeutic outcome in comparison with both non-targeted complex and free drug. The diagnostic capability of the prepared platform was also evaluated implementing C26-tumor-bearing mice. Obtained data confirmed higher tumor accumulation and higher tumor residence time for targeted complex through MRI imaging due to the existence of SPION as a contrast agent in the core of the prepared complex. The prepared multimodal theranostic system provides a safe and effective platform for fighting against cancer.

A DNA nanosensor for monitoring ligand-induced i-motif formation.

Herein, we present a gold nanoparticle (GNP)-based DNA nanosensor to detect the formation of an i-motif from the random coil structure by small molecules at physiological pH. The nanosensor shows a distance dependent fluorescence turn-off response in the presence of a ligand, indicating conformational changes from the C-rich single stranded DNA into an i-motif.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Recent advances in functionalization of plasmonic nanostructures for optical sensing.

This review summarizes the progress that has been made in the use of nanostructured SPR-based chemical sensors and biosensors. Following an introduction into the field, a first large section covers principles of nanomaterial-based SPR sensing, mainly on methods using noble metal nanoparticles (spheres, cubes, triangular plates, etc.). The next section covers methods for functionalization of plasmonic nanostructures, with subsections on functionalization using (a) amino acids and proteins; (b) oligonucleotides, (c) organic polymers, and (d) organic compounds. Several tables are presented that give an overview on the wealth of methods and materials published. A concluding section summarizes the current status, addresses current challenges, and gives an outlook on potential future trends. This review is not intended to be a comprehensive compilation of the literature in the field but rather is a systematic overview of the state of the art in surface chemistry of plasmonic nanostructures. The ability of various ligands and receptors for functionalization of nanoparticles as well as their sensing capability is discussed.

Gold nanorods-based lateral flow biosensors for sensitive detection of nucleic acids.

A gold nanorod (AuNR)-based lateral flow nucleic acid biosensor (LFNAB) is reported for visual detection of DNA with a short test time and high sensitivity. AuNRs with an approximate length of 60 nm were utilized as a colored tag to label the detection DNA probe (Det-DNA). The capture DNA probe (Cap-DNA) was immobilized on the test region of LFNAB. Sandwich-type complex was formed among the AuNR-Det-DNA, target DNA (Tar-DNA), and Cap-DNA on the LFNAB by Watson-Crick base pairing. In the presence of Tar-DNA, AuNRs were thus seized on the test region of LFNAB, and the accumulation of AuNRs subsequently produced a characteristic colored band. The optimized LFNAB was able to detect 10 pM Tar-DNA without instrumentation. Quantitative analysis could be established by measuring the intensity of test band using a portable strip reader, and the detection limit of 2 pM target DNA was achieved on the LFNAB without signal amplification. The detection limit of the AuNR-based LFNAB is 250-fold lower than that of gold nanoparticle (AuNP)-based LFNABs. This work unveiled a sensitive, rapid, and economical strategy for the detection of nucleic acids, and simultaneously opening new promising routes for disease diagnosis and clinical applications. Gold nanorods are used as colored tags for lateral flow nucleic acid biosensor.

Fabrication of gold/silver nanodimer SERS probes for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R2 = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R2 = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.

Microfluidic particle accumulation for visual quantitation of copper ions.

A portable biosensor has been developed based on microfluidic particle accumulation for visual quantification of copper ions. A copper-dependent DNAzyme is used to connect magnetic microparticles (MMPs) and polystyrene microparticles (PMPs), forming "MMPs-DNAzyme-PMPs." When copper ions are present, the DNAzyme is cleaved, allowing free PMPs to be released from the MMPs-DNAzyme-PMP complex. Using a capillary-flow-based microfluidic device, the MMPs-DNAzyme-PMPs are first separated by a magnetic chamber, allowing the free PMPs to continue flowing until being trapped at a particle dam with a narrowing nozzle. Therefore, as a thermometer-like display, the copper level can be visually quantified by the accumulation length of the free PMPs in the trapping microchannel. The limit of detection (LOD) is 33 nM determined by the linear range of 25-100 nM, which is 900 times lower than the prevalent standard (~30 µM) in Hong Kong. The system shows excellent selectivity (> 1000-folds) against other heavy metal ions and abilities to adapt to multiple water environmental conditions. Tests on tap water samples and three local natural water sources in Hong Kong manifest that the device can effectively monitor the quality of freshwater with >70% recovery and 26.16% RSD.

A disposable gold foil paper-based aptasensor for detection of enteropathogenic Escherichia coli with SERS analysis and magnetic separation technology.

Rapid and sensitive detection of enteropathogenic Escherichia coli (EPEC) in fluids with complex background is an important task for safety quality control in the field of medicine, environment, and food. In this study, a gold foil paper-based aptasensor was developed for the detection of enteropathogenic EPEC O26:K60 with surface-enhanced Raman spectroscopy (SERS) and magnetic separation technology mediated by Fe3O4@Au composite. The gold foil paper was firstly modified with thiolated capture probe and SERS tag. The thiolated aptamer probe for EPEC was immobilized onto a Fe3O4@Au composite. In the presence of EPEC, highly specific recognition between the aptamer probe and EPEC made the Fe3O4@Au composite partially dissociated from the gold foil paper. This led to a decreased Raman intensity response, which showed an obvious negative linear correlation with increasing concentration of EPEC over a wide concentration range from 10 to 107 CFU/mL under an excitation wavelength of 633 nm. The detection limit was about 2.86 CFU/mL in a buffer solution and a licorice extractum and the detection time was only 2.5 h. The results demonstrate that the gold foil paper-based aptasensor can be an excellent biosensing platform that offers a reliable, rapid, and sensitive alternative for EPEC detection.

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP).

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection.

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)-based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 µM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 µM vs. 2.7 µM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions.

Impedimetric determination of cortisol using screen-printed electrode with aptamer-modified magnetic beads.

A non-invasive aptamer-based electrochemical biosensor using disposable screen-printed graphene electrodes (SPGEs) was developed for simple, rapid, and sensitive determination of cortisol levels. Selective detection of cortisol based on a label-free electrochemical assay was achieved by specific recognition of the cortisol DNA aptamer (CApt). The CApt was modified with streptavidin magnetic beads (MBs) before simple immobilization onto the electrode surface using a neodymium magnet. The electrochemical behavior of the aptamer-based biosensor was assessed by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) (vs Ag/AgCl). The specific binding between cortisol and CApt resulted in a decrease in charge transfer resistance (Rct) from EIS using [Fe(CN)6]3-/4- with increasing cortisol concentration. Under optimal conditions, a linear range from 0.10 to 100 ng/mL with a low detection limit (3SD/slope) of 2.1 pg/mL was obtained. Furthermore, the proposed biosensing system exhibited a satisfactory recovery in the range 97.4-109.2% with 5.7-6.6% RSD in spiked artificial human sweat. Regarding the applications of this tool, the aptamer-based biosensor has potential to be a versatile and point-of-care (POC) device for simple, sensitive, selective, disposable, and low-cost cortisol detection.

DNAzyme-Au nanoprobe coupled with graphene-oxide-loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase.

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu2+ ions. In the presence of ALP, the released Cu2+ can promote the Cu2+-dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L-1, and the limit of detection (LOD) is about 0.14 U L-1. The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA.

A reagent-less DNA sensor has been developed exploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87 nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10 min, in both cases. Amplification time was further optimized to be 40 min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.

Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors.

The biology of the late twentieth century was marked by the discovery in 1993 of a new class of small non-coding ribonucleic acids (RNAs) which play major roles in regulating the translation and degradation of messenger RNAs. These small RNAs (18-25 nucleotides), called microRNAs (miRNAs), are implied in several biological processes such as differentiation, metabolic homeostasis, or cellular apoptosis and proliferation. The discovery in 2008 that the presence of miRNAs in body fluids could be correlated with cancer (prostate, breast, colon, lung, etc.) or other diseases (diabetes, heart diseases, etc.) has made them new key players as biomarkers. Therefore, miRNA detection is of considerable significance in both disease diagnosis and in the study of miRNA function. Until these days, more than 1200 miRNAs have been identified. However, traditional methods developed for conventional DNA does not apply satisfactorily for miRNA, in particular due to the low expression level of these miRNA in biofluids, and because they are very short strands. Electrochemical biosensors can provide this sensitivity and also offer the advantages of mass fabrication, low-cost, and potential decentralized analysis, which has wide application for microRNAs sensing, with many promising results already reported. The present review summarizes some newly developed electrochemical miRNA detection methods.

Electrochemiluminescence from a biocatalysis accelerated N-(aminobutyl)-N-(ethylisoluminol)/dissolved O2 system for microRNA detection.

A kind of biocatalyst, laccase, has been employed as a biocompatible coreactant accelerator to efficiently catalyze coreactant (dissolved O2) for generating high local concentration of superoxide radical (O2•-), acquiring high-intense electrochemiluminescence (ECL) emission of ABEI (N-(aminobutyl)-N-(ethylisoluminol))/dissolved O2 system. Furthermore, a modified strand displacement reaction with excellent amplification efficiency was constructed by replacing traditional single strand DNA to the hairpin DNA as template for triggering the immobilization of more signal probes. As a result, the biosensor for microRNA-21 determination has preeminent selectivity and favorable sensitivity with detection limit down to 80.8 aM. Significantly, the devised strategy has blazed a new path for seeking more coreaction accelerators with splendid biocompatibility thus promoting the application of ternary ECL systems in biological analysis and clinical diagnosis.