Amphiphile-Mediated Ultrasmall Aggregation Induced Emission Dots for Ultrasensitive Fluorescence Biosensing

The development of ultrasensitive and highly selective fluorescence biosensors for diverse analytes is highly desirable but remains a challenge. It is attributable to the scarcity of fluorogens with promising brightness, stability, and nontoxicity, which primarily determine the performance of fluorescence biosensors. Herein, we report the design and preparation of aggregation induced emission (AIE) dots with high brightness, exceptional colloidal stability, ultrasmall size, and functional groups for developing ultrasensitive biosensor through the electrostatic conjugation to biological molecules, and use blemycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of the quencher-labeled DNA (Q-DNA) by BLM result in the formation of three-mer quencher-linked oligonucleotide fragments (Q-DNA-1), which significantly decreases the amount of quenchers anchored on AIE dot surfaces and subsequently reduces the fluorescence resonance energy transfer (FRET) effect. As compared to the case in which BLM is absent, remarkable fluorescence enhancement is observed, and is dependent on BLM concentration. Thus, ultrasensitive fluorescence detection of target BLM is realized, with a detection limit down to 3.4 fM, the lowest value reported so far. Moreover, the proposed fluorescence biosensor has also been successfully utilized for detection of BLM spiked in human serum samples. The as-proposed strategy not only significantly improves the selectivity and sensitivity of BLM assay, but also allows the ultrasensitive detection of a variety of bioactive molecules by simply changing the specific target recognition substances, thus providing a versatile fluorescence platform, and showing great potential to be applied in chemo-/bioanalysis and clinical biomedicine

HRP-Mimicking DNAzyme-Catalyzed in Situ Generation of Polyaniline To Assist Signal Amplification for Ultrasensitive Surface Plasmon Resonance Biosensing

It is well-known that the horseradish peroxidase- (HRP-) mimicking DNAzyme, namely, hemin/G-quadruplex, can effectively catalyze the polymerization of aniline to form DNA-guided polyaniline. Meanwhile, polyaniline exhibits extraordinary electrical, electrochemical, and redox properties, as well as excellent SPR signal-enhancing ability. Herein, we report a novel ultrasensitive surface plasmon resonance (SPR) biosensor based on HRP-mimicking DNAzyme-catalyzed in situ formation of polyaniline for signal amplification, using bleomycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of DNA probe P1 by BLM switches off the hybridization between P1 and the G-rich DNA probe P2, resulting in less hemin/G-quadruplex complexes and reduced DNA-guided polyaniline deposition on the SPR Au disk surface. As compared to the case when BLM is absent, a significant shift in SPR angle is observed, which is dependent on the BLM concentration. Therefore, ultrasensitive SPR detection of the target BLM is realized, with a detection limit down to 0.35 pM, much lower than those reported in the literature. Moreover, the proposed SPR biosensor has been successfully applied for the detection of BLM spiked in human serum samples. The HRP-mimicking DNAzyme-catalyzed in situ polyaniline deposition and polyaniline-assisted signal amplification not only significantly improves the specificity and the sensitivity of the BLM assay but also allows the ultrasensitive detection of other biomolecules by simply changing the specific target recognition DNA sequences, thus providing a versatile SPR biosensing platform for the ultrasensitive detection of a variety of analytes and showing great potential for application in the fields of bioanalysis and clinical biomedicine

Label-Free and Ultrasensitive Biomolecule Detection Based on Aggregation Induced Emission Fluorogen via Target-Triggered Hemin/G-Quadruplex-Catalyzed Oxidation Reaction

Fluorescence biosensing strategy has drawn substantial attention due to their advantages of simplicity, convenience, sensitivity, and selectivity, but unsatisfactory structure stability, low fluorescence quantum yield, high cost of labeling, and strict reaction conditions associated with current fluorescence methods severely prohibit their potential application. To address these challenges, we herein propose an ultrasensitive label-free fluorescence biosensor by integrating hemin/G-quadruplex-catalyzed oxidation reaction with aggregation induced emission (AIE) fluorogen-based system. l-Cysteine/TPE-M, which is carefully and elaborately designed and developed, obviously contributes to strong fluorescence emission. In the presence of G-rich DNA along with K⁺ and hemin, efficient destruction of l-cysteine occurs due to hemin/G-quadruplex-catalyzed oxidation reactions. As a result, highly sensitive fluorescence detection of G-rich DNA is readily realized, with a detection limit down to 33 pM. As a validation for the further development of the proposed strategy, we also successfully construct ultrasensitive platforms for microRNA by incorporating the l-cysteine/TPE-M system with target-triggered cyclic amplification reaction. Thus, this proposed strategy is anticipated to find use in basic biochemical research and clinical diagnosis

Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA Biosensing via Target-Triggered Ru(III) Release and Redox Recycling

A new label-free and enzyme-free ratiometric homogeneous electrochemical microRNA biosensing platform was constructed via target-triggered Ru­(III) release and redox recycling. To design the effective ratiometric dual-signal strategy, [Ru­(NH₃)₆]³⁺ (Ru­(III)), as one of the electroactive probes, was ingeniously entrapped in the pores of the positively charged mesoporous silica nanoparticle (PMSN), and another electroactive probe, [Fe­(CN)₆]^3– (Fe­(III)), was selected to facilitate Ru­(III) redox recycling due to its distinctly separated reduction potential and different redox properties. Owing to the liberation of the formed RNA–ssDNA complex from PMSN, the target miRNA triggered the Ru­(III) release and was quickly electroreduced to Ru­(II), and then, the in-site-generated Ru­(II) could be chemically oxidized back to Ru­(III) by Fe­(III). Thus, with the release of Ru­(III) and the consumption of Fe­(III), a significant enhancement for the ratio of electroreduction current [Ru­(NH₃)₆]³⁺ over [Fe­(CN)₆]^3– (<i>I</i>_Ru(III)/<i>I</i>_Fe(III)) value was observed, which was dependent on the concentration of the target miRNA. Consequently, a simple, accurate, and ultrasensitive method for the miRNA assay was readily realized. Furthermore, the limit of detection (LOD) of our method was down to 33 aM (S/N = 3), comparable or even superior to other approaches reported in literature. More importantly, it also exhibited excellent analytical performance in the complex biological matrix cell lysates. Therefore, this homogeneous biosensing strategy not only provides an ingenious idea for realizing simple, rapid, reliable, and ultrasensitive bioassays but also has a great potential to be adopted as a powerful tool for precision medicine

A: The dorsal view of DaZao 5^th instar larvae.

<p>B: The expression profile of <i>BmPAH</i> mRNA in the integument of 4^th instar larvae during molting from 0 to 24 h. <i>BmActin3</i> gene was used as an internal control.</p

Enzymatic Fuel Cell-Based Self-Powered Homogeneous Immunosensing Platform via Target-Induced Glucose Release: An Appealing Alternative Strategy for Turn-On Melamine Assay

Enzymatic fuel cell (EFC)-based self-powered biosensors have attracted considerable attention because of their unique feature of no need for extra power sources during the entire detection process, which endows them with the merits of simplicity, rapidness, low cost, anti-interference, and ease of use. Herein, we proposed, for the first time, an EFC-based self-powered homogeneous immunosensing platform by integrating the target-induced biofuel release and bioconjugate immunoassay for ultrasensitive melamine (ME) detection. In this design, the biofuel, i.e., glucose molecules, was entrapped in the pores of positively charged mesoporous silica nanoparticles and capped by the biogate AuNPs-labeled anti-ME antibody (AuNPs-Ab). The presence of the target ME triggered the entrapped glucose release due to the removal of the biogate via immunoreaction, which resulted in the transfer of electrons produced by glucose oxidation at the bioanode to the biocathode, and thus, the open-circuit voltage of the EFC-based self-powered immunosensor dramatically increased, realizing the ultrasensitive turn-on assay for ME. The limit of detection for ME assay was down to 2.1 pM (S/N = 3), superior to those previously reported in the literature. Notably, real milk samples need no special sample pretreatment for the detection of ME because of the good anti-interference ability of EFC-based self-powered biosensors and the excellent selectivity of the homogeneous immunoassay. Therefore, this appealing self-powered homogeneous immunosensing platform holds great promise as a successful prototype of portable and on-site bioassay in the field of food safety

The structure of <i>BmPAH</i> gene and identification of BmPAH expressed <i>in vitro</i>.

<p>A: The structure of the <i>BmPAH</i> gene. Exons are represented by boxes, with the number indicating the length of individual exon; the length of the introns is also shown. B: 12% SDS-PAGE of proteins stained with Coomassie brilliant blue. A protein of approximately 52 kDa was recognized. lane 1: protein standard; lane 2: 0.3 µg purified protein; lane 3: 0.6 µg purified protein. C: Peptide mass fingerprint by MALDI-TOF-MS.</p

Detailed data for <i>BmPAH</i> RNAi of GaoBai eggs.

<p>Detailed data for <i>BmPAH</i> RNAi of GaoBai eggs.</p

The phenotype of discolored markings (eye-shaped markings, semilunar markings, star spots).

<p>Red arrow indicates discolored markings after esculetin injection. Bar represents 1 mm.</p

Effects of <i>BmPAH</i> RNAi on GaoBai neonatal larvae plotted as mean ± SD (n = 3).

<p>A: The relative expression level of <i>BmPAH</i> mRNA by quantitative RT-PCR. Eukaryotic translation initiation factor 4A (silkworm microarray probe ID: sw22934) was used as an internal control. dsRNA-1 and dsRNA-2 induced unsuccessful coloring or normal coloring in neonatal larvae after injection of eggs with dsRNA-1 and dsRNA-2. B: Concentration of tyrosine from neonate larvae. Unsuccessfully colored samples were from unsuccessfully colored neonatal larvae arising from eggs injected with dsRNA-1 and dsRNA-2, and normal coloring samples were from normally colored neonatal larvae arising from eggs injected with dsRNA-1 and dsRNA-2. * represents significant differences at P<0.05; ** represents significant differences at P<0.01.</p