High content design of riboswitch biosensors: All-around rational module-by-module design.

Riboswitches are ideal, diverse regulatory elements with simple mechanisms. Therefore, they are widely employed in synthetic biology to construct highly efficient biosensors for biosensing and bioimaging. This review presents a rational riboswitch biosensor design that combines computational and hybrid methods. Four main steps are discussed, including the rational design of riboswitch screening libraries, rational riboswitch screening, rational tailoring, and the rational selection of reporting elements. Furthermore, the application of riboswitch biosensors for molecular detection and biological imaging is introduced. Finally, the future development and construction of ideal riboswitch biosensors are discussed, while assessing their application for point-of-care testing, health monitoring, and gene therapy. This review is expected to provide a comprehensive overview of the rational construction of riboswitch biosensors.

Sandwich capture ultrasensitive sensor based on biohybrid interface for the detection of Cronobacter sakazakii.

A simple, rapid and ultrasensitive visual sensing method for the detection of Cronobacter sakazakii (C. sakazakii) based on a biohybrid interface was established. During the entire sensing process, quadruple-cascade amplification showed its superior sensing performance. First, the prepared immunomagnetic beads (IMB) were used to isolate and enrich specific targets from the food matrix. After adding the fusion aptamer, the aptamer sequence specifically recognized the target and formed the immune sandwich structure of antibody-target-fusion aptamer. In addition, the fusion aptamer also included the template sequence of exponential amplification reaction (EXPAR), which contained the antisense sequence of the G-rich sequence. Therefore, a large number of G-rich sequences can be generated after EXPAR can be triggered in the presence of Bst. DNA polymerase, nicking endonuclease, cDNA, and dNTP. They were self-assembled into G-quadruplex structures and then combined with hemin to form G4/hemin DNAzyme, resulting in visible coloration and measuring absorbance at 450 nm for quantitative detection. The assay showed a limit of detection (LOD) of 2 CFU/mL in pure culture and 12 CFU/g in milk powder in optimal conditions. This method provides a promising strategy for rapid and point-of-care testing (POCT) since it does not require DNA extraction, medium culturing, and expensive instrumentation. KEY POINTS: •Single-cell level detection of C. sakazakii with ultrasensitive and rapidness •The fusion aptamer integrated recognition and amplification •Sensing analysis of C. sakazakii based on cascade amplification of biohybrid interface.

Rapid label-free colorimetric dual-functional aptasensor for ß-lactoglobulin detection based on a rational tailoring strategy.

ß-lactoglobulin (ß-LG) is a nonnegligible allergenic protein found in cow milk. A label-free, enzyme-free, dual-functional aptameric sensor was constructed based on rational aptamer tailoring for ß-LG detection. Via an established three-step rational tailoring strategy, the original aptamer was transformed from an inflexible antiparallel topology into the flexible, antiparallel/parallel hybrid topology, enlg2-pl3. enlg2-pl3 displayed the dual-functionality of not only binding with ß-LG via the antiparallel conformation but also outputting signals via the parallel type. The dual-functionality depended on the conformational conversion of enlg2-pl3. The ß-LG-driven conformational conversion to antiparallel type was the detection basis of aptasensor. The detection conditions were optimized regarding ions, hemin, chromogenic substrate, pH levels, and detection time. Furthermore, the aptasensor offered advantages, such as cost-efficiency, simple operation, 40 min rapid detection, excellent specificity, and a sensitivity of a three-orders of magnitude linear range and an 8.23-fold linear curve slope, meeting the point-of-care testing (POCT) requirements for dairy analysis.

Functional nucleic acid lateral flow magnetic biosensor based on blocking the super PCR and magnetic test strip for rapid detection of genetically modified maize MON810†.

Although PCR is a typical temperature-variable amplification technique for nucleic acids, it still faces challenges in rapid screening, such as low speed, poor sensitivity, inferior visualization, and weak stability. In this paper, a universal functional nucleic acid (FNA) lateral flow magnetic biosensor was constructed using blocking super PCR (BS-PCR) and a magnetic test strip (MTS). In theory, the visualized and magnetic output of dsDNA-based amplicons were achieved via ssDNA/dsDNA conversion by blocking linkers in the PCR primers and magnetic probes. In application, high-speed, super-sensitive, highly stable rapid screening was realized by taking advantage of the speediness of the super PCR reactor and the anti-background interference, visualization and high stability of magnetic signal readout. The genetically modified maize MON810 was selected as a double-stranded model target, while 5-min BS-PCR and 5-min magnetic signal readouts achieved the screening results within 10 min. Furthermore, the exponential PCR amplification and magnetic-based sensibilization improved the sensitivity to a single copy. This biosensor is simple and portable, with significant potential for rapid on-site screening.

MSN/NA-doped nanoflower enhancing isothermal fluorescent sensor with a portable PCR tube fluorescence reader for the on-site detection of Vibrio parahaemolyticus.

To develop an on-site, thermostatic and rapid sensor for the detection of Vibrio parahaemolyticus (V. parahaemolyticus), A single cross-priming fluorescence (SCPF) sensor was designed using a 3D nano-nucleic acid hybrid material that termed mesoporous silica nanoparticle/nucleic acid-doped nanoflower (MSN/NA-doped nanoflower). In addition, a portable polymerase chain reaction (PCR) tube fluorescence reader was built. Further analysis of the MSN/NA-doped nanoflower morphology and the enhancement mechanism indicated that the MSNs aggregated into larger nanoclusters by adsorbing single cross-priming amplification (sCPA) components, forming MSNs/NAs-doped nanoflowers, and increasing the local concentrations to enhance sCPA efficiency. Cyclic amplification relied mainly on the self-folding hairpin-like structure of the amplified product that continuously formed, opened, re-formed, and opened again. The target DNA was detected with a detection limit of 2.4 copies/µL.

Insights into nucleic acid-based self-assembling nanocarriers for targeted drug delivery and controlled drug release.

Over the past few decades, rapid advances of nucleic acid nanotechnology always drive the development of nanoassemblies with programmable design, powerful functionality, excellent biocompatibility and outstanding biosafety. Nowadays, nucleic acid-based self-assembling nanocarriers (NASNs) play an increasingly greater role in the research and development in biomedical studies, particularly in drug delivery, release and targeting. In this review, NASNs are systematically summarized the strategies cooperated with their broad applications in drug delivery. We first discuss the self-assembling methods of nanocarriers comprised of DNA, RNA and composite materials, and summarize various categories of targeting media, including aptamers, small molecule ligands and proteins. Furthermore, drug release strategies by smart-responding multiple kinds of stimuli are explained, and various applications of NASNs in drug delivery are discussed, including protein drugs, nucleic acid drugs, small molecule drugs and nanodrugs. Lastly, we propose limitations and potential of NASNs in the future development, and expect that NASNs enable facilitate the development of new-generation drug vectors to assist in solving the growing demands on disease diagnosis and therapy or other biomedicine-related applications in the real world.

Fusion of binary split allosteric aptasensor for the ultra-sensitive and super-rapid detection of malachite green.

The complicated labeling procedure and high cost of split aptasensors have hitherto limited their application in the detection of hazardous substances. Herein we report the first examples of label-free aptasensors based on the fusion of a binary split G-quadruplex and malachite green (MG) aptamer, transducing recognition events into fluorescent signals through the allosteric regulation of the aptamer to achieve selective and sensitive detection. Specifically, RNA MGA was successfully converted into DNA MGA with comparable affinity and improved stability, thereby overcoming the limitations of poor stability and high expense. We subsequently split the DNA MGA and attached them to a G-rich DNA sequence at the 5' and 3' ends, to construct the binary split allosteric aptasensor. The performance of the binary split aptasensor for MG detection was significantly improved based on proposed allosteric regulation strategy, and the reconfiguration capability of the aptamers upon binding with targets was proven, providing the binary split aptasensor with superior sensitivity and selectivity. This sensing method has a wide dynamic detection range of 5 nmol·L-1 to 500 µmol·L-1, with a low limit of detection (LOD) of 4.17 nmol·L-1, and achieves the ultra-sensitive and super-rapid detection of MG. This newly proposed aptasensor is equipped with the advantages of being label-free, time saving and economical. More importantly, this successful construction of a fused aptasensor expands the principles of split aptasensor design and provides a universal platform for the detection of environmental contaminants.

Assembly and in vitro assessment of a powerful combination: aptamer-modified exosomes combined with gold nanorods for effective photothermal therapy.

Due to good biocompatibility and plasma membrane similarity, the nanosized exosomes are ideal drug carriers. Near-infrared (NIR) photothermal therapy is an emerging method for cancer treatment in which photothermal agents absorb the energy of external NIR light to generate high temperatures in a targeted region to effectively kill cancer cells. Gold nanorods (AuNRs) have been found to provide a prominent photothermal performance, while aptamers can precisely target surface markers on cells with high affinity and specificity. In this study, exosomes were mildly functionalized by integrating them with aptamers and AuNRs to assemble a powerful combination Apt-Exos-AuNRs (AEARs) with good specificity and an effective photothermal killing action on cancer cells. The structure, hydrodynamic diameters, zeta potential, UV-vis absorption spectra and stability of the AEARs were further characterized. In addition, using a cell model, the cancer cell targeting ability of the AEARs and its cellular uptake were observed. Moreover, its photothermal killing effect on various human cancer cells in vitro was validated by a CCK-8 assay as well as apoptosis analysis, the results of which suggest this exosomes-based nanomaterial can serve as a novel and broad-spectrum platform for precision cancer therapy.

Duplex-specific nuclease-resistant triple-helix DNA nanoswitch for single-base differentiation of miRNA in lung cancer cells.

In this work, a duplex-specific nuclease (DSN)-resistant triplex-helix DNA nanoswitch was designed for assays of single-base differentiation of the let-7a family in lung cancer cells. Initially, although a 10-bp duplex stem in the nanoswitch was cleaved to pieces, a 10-bp triplex stem was resistant to DSN. Consequently, a triple-stranded DNA structure resistant to DSN was obtained. The pH-dependent formation of the triplex structure then produced the pH-related nanoswitch/miRNA hybrid, and the metastable nanoswitch generated an obvious signal increase at pH 6.8. Surprisingly, the pH condition at 6.8 for the best nanoswitch/miRNA hybrid is consistent with the optimal DSN catalysis, which paves the way for a first-rank DSN signal amplification (DSNSA) strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM. The cyclic strategy based on the DSN-mediated triplex-helix DNA nanoswitch was verified in lung cancer cell samples and exhibited better discriminatory ability without user-unfriendly nucleotide modification or extra probe-mediated assistance, showing excellent potential for application in biomedical sensing and clinical diagnosis. Graphical abstract Based on the discovery that a triple-helix DNA nanoswitch is resistant to DSN and that the nanoswitch/miRNA hybridization was pH-related, pH at 6.8, which is suitable for the optimal nanoswitch/miRNA hybrid and DSN catalysis, reinforced the DSNSA strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM.

Rapid strand replacement primer thermostat visual sensor based on Bst DNA polymerase and pyrophosphatase for detecting Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major hidden danger of food safety. To develop a rapid, sensitive and on-site detecting method of Vibrio parahaemolyticus (V. parahaemolyticus), a strand replacement primer thermostat phosphate (SRPP) visual sensor was proposed, based on Bst DNA polymerase and pyrophosphatase. The novel strand replacement primer (SRP) facilitates chain substitution and to open a self-folding hairpin by adding region at its 3' end. Under the action of the SRP, a pair of external primers and two inner primers, target DNA is specifically amplified at 63 °C relies mainly on the hairpin. Many pyrophosphates (PPi) are simultaneously generated as by-products, which can be converted into phosphates (Pi) by pyrophosphatase for phosphomolybdate blue visual detection within 5 min. The proposed biosensor can detect 1.29 × 103 copies of V. parahaemolyticus within 35 min.

TiO2 Nanoparticle-Enhanced Linker Recombinant Strand Displacement Amplification (LRSDA) for Universal Label-Free Visual Bioassays.

The influence of nanomaterials on dynamic isothermal amplification and their morphology regulated by bionic biological reactions in vitro remain unknown. From a theoretical perspective, TiO2 nanoparticles enhance the amplification efficiency and reaction specificity of recombinase polymerase amplification (RPA). These nanoparticles aggregated into larger nanoclusters by adsorbing RPA components, termed nanoscale RPA factories, which increased their local concentrations to enhance RPA. Following the nick/extension cycles mediated by a bifunctional linker located at the 5' end of the forward primers, the TiO2 nanoparticle-enhanced LRSDA process produces single-stranded products, constituting the G-quadruplex DNAzymes and catalyzing the chromogenic substrate to facilitate colorimetric analysis for on-site bioassays. Salmonella spp. and genetically modified maize MON810 could be detected with a detection limit of 4 cfu/mL and 0.1% transgenic components, respectively. Briefly, TiO2-assisted isothermal molecular amplification addressed the demands of practical on-site applications.

Physiological changes in Chlamydomonas reinhardtii after 1000 generations of selection of cadmium exposure at environmentally relevant concentrations.

Cadmium (Cd) is a nonessential and toxic trace element widely existing in waters through various anthropogenic activities such as mining and waste disposal. The physiological responses of aquatic organisms to long-term Cd exposure at environmentally relevant concentrations are still not well explored. In the present study, two strains of unicellular green algae Chlamydomonas reinhardtii, a walled strain CC125 and a wall-less strain CC406 were selected to investigate the physiological changes of aquatic organisms after long-term Cd exposure at environmentally relevant concentrations (4.92 and 49.2 µg L-1). After about 1000 generations of selection, all of the two strains showed higher intracellular lipid peroxidation and lower photosynthetic activities, and failed to evolve specific adaptation to high levels of Cd (4.92 mg L-1) compared to the control. However, short-term low dose Cd exposure exerted hormetic effects on C. reinhardtii and the hormetic stimulation of growth rate, chlorophyll contents and photochemical activities at the lower concentration of Cd (4.92 µg L-1) groups were more pronounced than those at higher ones (49.2 µg L-1). Taken together, this study confirmed that long-term exposure to Cd at environmentally relevant concentrations which were regarded as nontoxic in acute experiments would produce toxic effects on C. reinhardtii and should be paid more attention.