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Aptamers are short and single-stranded DNA or RNA molecules with highly programmable structures that give them the ability to interact specifically with a large variety of targets, including proteins, cells, and small molecules. Multivalent aptamers refer to molecular constructs that combine two or more identical or different types of aptamers. Multivalency increases the avidity of aptamers, a particularly advantageous feature that allows for significantly increased binding affinities in comparison with aptamer monomers. Another advantage of multivalency is increased aptamer stabilities that confer improved performances under physiological conditions for various applications in clinical settings. The current study aims to review the most recent developments in multivalent aptamer research. The review will first discuss structures of multivalent aptamers. This is followed by detailed discussions on design strategies of multivalent aptamer approaches. Finally, recent developments of the multivalent aptamer approach in biosensing and biomedical applications are highlighted. [ABSTRACT FROM AUTHOR]

Aptamers have been widely used to recognize and capture their targets in sensitive detection applications, such as in detections of circulating tumor cells. In this study, we investigate the effects of different lengths of oligo-T spacers on surface tethered sgc8 aptamers and their target capturing performances. To achieve this, sgc8 aptamers were immobilized onto microfluidic channel surfaces via oligo-T spacers of different lengths, and the target capturing performances of these immobilized aptamers were studied using CCRF-CEM cells. We demonstrate that the capturing performances of the immobilized aptamers were significantly affected by steric hindrance. Our results also show that aptamers immobilized on surfaces via spacers of ten Ts demonstrated the best cell capturing performances; aptamers with either too short or too long oligo-T spacers showed reduced cell capturing performances. Therefore it can be concluded that spacer optimizations are critically important for surface tethered aptamers that are commonly used in microfluidic devices for sensitive target sensing and detections. [ABSTRACT FROM AUTHOR]

The majority of foodborne illnesses and deaths are caused by pathogenic bacteria. Therefore there is an increasingly urgent need for rapid, sensitive and cost-effective detection methods in order to avoid or to respond to outbreaks of such diseases in a timely manner. In an attempt to develop a simple and sensitive fluorescence intensity based microfluidic detection platform, PAMAM dendrimer was immobilized onto PDMS microchannel that was further modified with DNA aptamers for detections of E. coli O157:H7 cells. To further improve detection performance, rolling circle amplification (RCA) was employed to enhance the fluorescence signals. In addition, RCA products were characterized by both agarose gel electrophoresis and atomic force microscopy. Our results showed that RCA was able to enhance detection signals by up to 50 times and that the limit of detection of the system was reduced to 10 2 cells/mL with excellent detection specificities. Therefore, it can be concluded that this simple dendrimer-aptamer based microfluidic detection platform with RCA signal enhancement is a promising method for rapid and sensitive detection for foodborne pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Aptamer based microfluidic platforms have been developed rapidly in recent years, and strategies to improve detection sensitivities of such platforms have attracted a significant amount of attention. To achieve whole cell sensitive detections by microfluidic devices, a new dual-rolling circle amplification (RCA) detection approach is presented in this study. This dual-RCA approach includes a capturing RCA (cRCA) reaction that is designed to modify microfluidic channel surfaces with long tandem repeating aptamers (i.e. poly-aptamers) to effectively capture target E. coli O157:H7 cells. We demonstrate that this poly-aptamers modified microchannels capture 3-fold more target cells in comparison with microchannels modified with mono-aptamers against the target cells. In addition, signalling RCA (sRCA) is employed in the dual-RCA design to further enhance detection signals. Our results show that the detection signals are enhanced by up to 50 times by sRCA when compared with those with single fluorescence probes. Furthermore, by combing both the cRCA and the sRCA in one dual-RCA detection system, we demonstrate that the detection signals can be significantly enhanced by ∼250-fold. We also show that E. coli O157:H7 detections with the dual-RCA approach can be used in different food matrices, including orange juice and milk where the limit of detection of 80 cells/mL is achieved. In conclusion, this microfluidic device in combination with a dual-RCA to enhance both target capturing and detection signals is a simple and promising approach to sensitive whole-cell detections for food safety inspections. Image 1 • A novel dual-RCA whole cell detection system is developed for rapid and sensitive detection applications using microfluidic devices. • A cRCA can significantly enhance target capturing, and a sRCA can significantly amplify detection signals. • The dual-RCA increases overall detection signal intensities by approximately 250 times when compared with detections without RCAs. • The dual-RCA whole cell detection achieves an excellent LOD of 80 cell/mL. [ABSTRACT FROM AUTHOR]

Abstract A microfluidic system that incorporates both dendrimers and aptamers to detect E. coli O157:H7 is developed. To achieve this, generation 7-polyamidoamine dendrimers were immobilized onto the detection surfaces of PDMS microfluidic channels; subsequently aptamers against E. coli O157:H7 were conjugated onto the microchannel surfaces via the immobilized dendrimers as templates. Surface modifications were characterized by FTIR, XPS, water contact angles, fluorescence microscopy and AFM to confirm the success of each surface modification steps. The efficacy of this simple microchannel in detection was investigated using E. coli O157:H7 spiked samples. Our results showed that this interesting approach significantly increased the amount of aptamers available on the microfluidic channel surfaces to capture E. coli O157:H7 cells to allow sensitive detection, which in turn resulted in detections of E. coli O157:H7 cells at a low limit of detection of 102 cells mL−1. The results also demonstrated that in comparison with the generation 4-polyamidoamine dendrimers (G4) modified microchannels, those modified with G7 showed enhanced detection signals, improved target capturing efficiencies, and at higher throughput. This simple whole cell detection design has not been reported in the literature and it is an interesting and effective approach to developing a sensitive and rapid detection platform for foodborne pathogenic bacteria. Graphical abstract Image 1 Highlights • A microfluidic system for sensitive detection of E. coli O157:H7 at a LOD of 102 cells mL-1 is developed. • Dendrimers are immobilized onto microfluidic channel surfaces to reduce background detection noise. • Dendrimer-aptamer modified surface provides multiple binding sites for enhanced target capturing at high throughput rates. • Detection performance of G7-aptamer and G4-aptamer modified microchannels are investigated and compared. • Surface modifications are characterized by FTIR, XPS, water contact angles, fluorescence microscopy and AFM. [ABSTRACT FROM AUTHOR]

We develop a multivalent aptamer meshed open pore membrane with a signal amplification method as a simple and robust platform for sensitive, robust, and high-flux detection of E. coli O157:H7 whole cells in food samples. Nylon open pore membranes are surface modified by multi-handled poly(amidoamine) dendrimers as templates that are subsequently functionalized with multiple branches of capturing RCA (cRCA). The resulting cRCA networks are composed of tandem repeating aptamers that can specifically recognize and capture E. coli O157:H7 cells. The captured cells are then conjugated with signaling RCA (sRCA) to amplify the fluorescent detection signal. Our results demonstrate that the cRCA-based aptamer meshwork significantly enhances the surface-capturing performance of E. coli O157:H7 cells while achieving high-fluxes. Moreover, the membrane surface modification using dendrimers renders the resulting membrane nonfouling thus significantly reducing the background noises by approximately 96 times while the sRCA signal intensification enhances the fluorescent detection signals by approximately 27 times. We show that the membranes exhibit excellent performances in detecting E.coli O157:H7 in milk and orange juice samples with limit of detection of 10 cells per 250 mL with excellent specificity, offering a robust and practical approach for rapid and sensitive detection of foodborne bacteria. • Membrane allows high flux detection of bacteria in complex food matrices. • Dendrimer surface modification decreases detection background noises. • Dendrimer multi-handle templates allow formation of aptamer network on the membrane. • cRCA products mesh the membrane open pores to enhance target capturing. • sRCA signal intensification amplifies detection signals for sensitive detection. [ABSTRACT FROM AUTHOR]

We developed a multi-pronged approach to enhance the detection sensitivity of localized surface plasmon resonance (LSPR) sensor chips to detect SARS-CoV-2. To this end, poly(amidoamine) dendrimers were immobilized onto the surface of LSPR sensor chips to serve as templates to further conjugate aptamers specific for SARS-CoV-2. The immobilized dendrimers were shown to reduce surface nonspecific adsorptions and increase capturing ligand density on the sensor chips, thereby improving detection sensitivity. To characterize the detection sensitivity of the surface-modified sensor chips, SARS-CoV-2 spike protein receptor-binding domain was detected using LSPR sensor chips with different surface modifications. The results showed that the dendrimer-aptamer modified LSPR sensor chip exhibited a limit of detection (LOD) of 21.9 pM, a sensitivity that was 9 times and 152 times more sensitive than the traditional aptamer- or antibody-based LSPR sensor chips, respectively. In addition, detection sensitivity was further improved by combining rolling circle amplification product and gold nanoparticles to further amplify the detection signals by increasing both the target mass and plasmonic coupling effects. Using pseudo SARS-CoV-2 viral particles as detection targets, we demonstrated that this combined signal intensification approach further enhanced the detection sensitivity by 10 folds with a remarkable LOD of 148 vp/mL, making it one of the most sensitive SARS-CoV-2 detection assays reported to date. These results highlight the potential of a novel LSPR-based detection platform for sensitive and rapid detection of COVID-19 infections, as well as other viral infections and point-of-care applications. [ABSTRACT FROM AUTHOR]

Poly(styrene) (PS) 96-well plates were surface modified to improve the detection performances of an otherwise traditional enzyme-linked immunosorbent assay (ELISA). Poly(amidoamine) generation 7 (G7) dendrimers were covalently immobilized on the surface of PS plates and subsequently conjugated with aptamers specific for a model analyte, i.e., human platelet-derived growth factor BB (PDGF-BB). This surface functionalization was followed by Fourier-transform infrared spectroscopy, water contact angle, atomic force microscopy, and X-ray photoelectron spectroscopy (XPS) to confirm the success of the modifications. Moreover, the assay performances of the G7-aptamer modified PS plates were compared to those of traditional ELISA performed on regular PS 96-well plates. The G7-aptamer assay demonstrated a 2.3-time broader linear detection range and a 13-time improved detection limit than the traditional ELISA. More importantly, the new G7-aptamer modified PS plates also showed excellent analytical specificities, detection recoveries, and precisions when the targets were assayed in a cell culture medium. This combined dendrimer templates and aptamers surface modification approach significantly reduces background noises and increases detection signals, and can be readily incorporated into existing ELISA workflows and many other PS microplate based high throughput and automated bioassays. [Display omitted] • A dendrimer-aptamer surface modification is developed to improve ELISA performances. • Dendrimers immobilized on PS surfaces reduce non-specific bindings. • Dendrimer templates allow multiple aptamer conjugations for enhanced target binding. • The new assay shows broader linear range and lowered LOD. [ABSTRACT FROM AUTHOR]

We investigated the application of rolling circle amplification (RCA) to modify microfluidic channels for potential sensitive detection applications. To this end, a novel in situ capturing RCA (cRCA) strategy was used to modify the inner surfaces of microfluidic channels with cRCA products that featured repeating tandem capturing aptamers specific for E. coli O157:H7 cells. We showed that the in situ cRCA reaction modified microfluidic channels demonstrated significantly enhanced capturing efficiency in a wide range of flow rates when compared with the unit-aptamer approach. We demonstrated for the first time that microfluidic surfaces modified with the in situ cRCA products showed peak capturing performances both in terms of target capturing efficiency and specificity, and this was likely due to unexpected base-pairing that resulted in altered secondary structures of the capturing aptamers. Our data suggest that the in situ cRCA surface modification is a promising strategy to improve capturing performances in microfluidic devices in sensitive detection applications that also require high throughput. However, cRCA reaction conditions, particularly reaction time and concentrations of initial circular template, must be carefully investigated before the potentials of the in situ cRCA surface modification approach can be fully realized. Image 1 • A capturing RCA (cRCA) approach is used to capture E. coli O157:H7 cells by microfluidic devices. • The cRCA approach renders capturing surfaces more efficient in capturing targets at higher flow rates. • The cRCA approach results in peak capturing performance due to unexpected base-pairing. • Capturing performance of the cRCA approach is evaluated in real-world food matrices. [ABSTRACT FROM AUTHOR]