A Sensitive and Homogeneous SNP Detection Using Cationic Conjugated Polymers

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. A SNP and allele frequency analysis method was established using an optically amplifying water-soluble cationic conjugated polymer and highly selective single base extension reaction in a simple, convenient, homogenous, and sensitive manner. The higher FRET efficiency between PFP and dGTP-Fl is correlated to the incorporation of dGTP-Fl into the probe by one base extension reaction when the target/probe pair is complementary at the polymorphic site. The mutant-type target DNA can be detected in the presence of the wild-type target in which the concentration is much higher than that of mutant-type target. As low as 2% allele frequency can be accurately determined by this new assay method

Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of <i>Plasmodium</i> in Asymptomatic Malaria

Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts

Flow Cytometry-Assisted Mix-and-Read Assay for Ultrasensitive Detection of Protein Kinase Activity by use of Zr⁴⁺-Functionalized Mesoporous SiO₂ Microspheres

A flow cytometry-assisted mix-and-read assay is developed for ultrasensitive detection of protein kinase activity by use of Zr⁴⁺-functionalized mesoporous SiO₂ microspheres (ZrMMs). This strategy integrates the distinct advantages of ZrMMs for highly specific recognition as well as high capacity binding of kinase-induced fluorescent phosphopeptides and flow cytometry for powerful and separation-free bead analysis, leading to an ultrahigh sensitivity for kinase analysis in a extremely simple mix-and-read manner. Furthermore, this ultrasensitive design is well suitable for detection of cell kinase activities in complex biological samples and for screening of potential protein kinase inhibitors, which is of great significance for the development of targeted therapy, clinical diagnosis, and studies of cellular signal transduction pathways

Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs’ population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests

Graphene Surface-Anchored Fluorescence Sensor for Sensitive Detection of MicroRNA Coupled with Enzyme-Free Signal Amplification of Hybridization Chain Reaction

A new enzyme-free signal amplification-based assay for microRNA (miRNA) detection is developed by using hybridization chain reaction (HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. MiRNAs can efficiently initiate HCR between two species of fluorescent hairpin probes. After HCR, both of the excess hairpin probes and the HCR products will be anchored on the GO surface. The fluorescence of the hairpin probes can be completely quenched by GO, whereas the HCR products maintain strong fluorescence. Therefore, integrating HCR strategy for signal amplification with selective fluorescence quenching effects of GO provides a versatile miRNA assay

General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection

Multivalent Engineering of Exosomes with Activatable Aptamer Probes for Specific Regulation and Monitoring of Cell Targeting

Reconstituting and probing exosome-cell interactions is critical for elucidating exosome-related cell biology and advancing their diagnostic and therapeutic potential. We report here an exosomal engineering strategy to achieve controlled regulation of exosome-cell interactions with activatable sensing capability. The approach relies on membrane-protein directed, programmable DNA self-assembly to construct a DNA polymeric scaffold with multivalent display of structure-switchable aptamer sensing probes on exosome surfaces. The engineered exosomes exhibit enhanced cancer cell targeting ability compared to exosomes modified with monovalent aptamers. Furthermore, the anchored aptamer probes could be activated by specific membrane protein targeting, followed by structural switching to report an output fluorescence signal, thus allowing dynamic monitoring of exosome-cell interactions both in vitro and in vivo. We envision this will provide a complementary tool for specific regulation and monitoring of exosome-cell docking interactions and will advance the development of exosome-based biomedical applications

Cationic Oligo(thiophene ethynylene) with Broad-Spectrum and High Antibacterial Efficiency under White Light and Specific Biocidal Activity against <i>S. aureus</i> in Dark

We designed and synthesized a novel oligo­(thiophene ethynylene) (OTE) to investigate the antibacterial activities against Gram-positive (<i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>) and Gram-negative (<i>Ralstonia solanacearum</i> and <i>Escherichia coli</i>) bacteria in vitro by photodynamic therapy (PDT). Notably, OTE presents broad-spectrum and greatly high antibacterial activities after white light irradiation at nanogram per milliliter concentrations. The half inhibitory concentrations (IC₅₀) values obtained for <i>S. aureus</i>, <i>S. epidermidis</i>, <i>E. coli</i>, and <i>R. solanacearum</i> are 8, 13, 24, and 52 ng/mL after illumination for 30 min, respectively, which are lower than that of other PDT agents. Interestingly, OTE shows the specific and very strong dark killing capability against <i>S. aureus</i> at the concentration of 180 ng/mL for 30 min, which is the highest efficiency biocide against <i>S. aureus</i> without the need of irradiation to date. The antibacterial mechanism investigated demonstrated that reactive oxygen species or singlet-oxygen generated by OTE kills bacteria irreversibly upon white light irradiation, and OTE as a v-type oligomer exerts its toxicity directly on destroying bacterial cytoplasmic membrane in the dark. Importantly, the OTE shows no cell cytotoxicity and excellent biocompatibility. The results indicate that it is potential to provide versatile applications in the efficient control of pathogenic organisms and specific application for killing <i>S. aureus</i>

Homogeneous and Sensitive Detection of microRNA with Ligase Chain Reaction and Lambda Exonuclease-Assisted Cationic Conjugated Polymer Biosensing

A simple and homogeneous microRNA assay is developed by integration of ligase chain reaction (LCR) and lambda exonuclease-assisted cationic conjugated polymer (CCP) biosensing. LCR is utilized for exponential amplification of microRNA, and lambda exonuclease is introduced to degrade excess fluorescein-labeled probes in LCR for eliminating background signal. After addition of CCP, efficient fluorescence resonance energy transfer from CCP to fluorescein in LCR products occurs. The method is sensitive enough to detect 0.1 fM target microRNA and specific to discriminate one-base difference of microRNAs, which paves a new way for homogeneous microRNA detection and molecular diagnosis

Binding Studies of Cationic Conjugated Polymers and DNA for Label-Free Fluorescent Biosensors

Cationic conjugated polymers (CCPs), especially polythiophene, have been extensively used as probes for developing DNA and aptamer-based biosensors. Although many interesting applications have been achieved, a fundamental understanding of this system remains quite limited. In this work, we performed systematic binding assays to understand the interactions between poly­(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene) (PMNT) and DNA. The fluorescence of PMNT at 530 nm initially decreased and then a peak at 580 nm emerged after binding with single-stranded DNA (ssDNA). The binding force between PMNT and DNA was dominated by electrostatic interactions at first and then DNA base-mediated interactions also became important. Since the bases in double-stranded DNA (dsDNA) were shielded, their fluorescence changes were quite different. To best differentiate ssDNA and dsDNA, the optimal pH was between 6 and 8, and the optimal NaCl concentration was around 0.3 M. Moreover, by changing the sequence and length of ssDNA, poly-T had the largest fluorescence shift and poly-A had the smallest change. Under the optimized conditions, the PMNT-based biosensor had a detection limit of 1 nM DNA, which was similar to the SYBR Green I-based assay