Controllable design of a nano-bio aptasensing interface based on tetrahedral framework nucleic acids in an integrated microfluidic platform

Highlights

• A tetrahedral framework nucleic acids-based aptasensing interface has been constructed in the microfluidic system.

• The development of an integrated microfluidic platform for bacterial detection, cell culture, and AST activities.

• A desirable detection sensitivity (as low as 10 CFU/mL) and specificity for E. coli O157: H7.

• The platform was able to provide fast (within 5 h) and reliable analysis of AST.

Abstract

The limited reaction time and sample volume in the confined space of microfluidic devices give considerable importance to the development of more effective biosensing interfaces. Herein, the self-assembling of tetrahedral framework nucleic acids (FNAs) with controllable size on the interface of the microfluidic microchannels is studied. Compared with macroscopic turbulence control on traditional micro-structured microfluidic surface, the novel FNA-engineered microfluidic interface successfully constructs a 3D reaction space at nanoscale by raising DNA probes away from the surface. This FNA interface dramatically improves the reaction kinetics during molecular recognition due to extremely ordered orientation, configuration and density of DNA probes on the surface. Finally, the FNA-engineered interface is applied in a novel multi-functional microfluidic platform, towards a “one-stop” assay of Escherichia coli O157: H7 (E. coli O157: H7), integrating capture, release, enrichment, cell culture and antimicrobial susceptibility testing (AST). With the FNA-aptamer probe, we achieved an enhanced bacterial detecting efficiency (10 CFU/mL) plus excellent selectivity and precision. The appicability was strongly demonstrated when the biosensor was successfully applied in real samples, including the analysis of antibiotic susceptibility and minimum inhibitory concentration (MIC) of E. coli O157: H7 among different antibiotics. The application of FNA interface will open a wide avenue for the development of microfluidic biosensors for other pathogenic microorganisms or circulating tumor cells (CTC) simply by changing the aptamers.

Introduction

For decades, microfluidic biosensors were widely applied in both basic and applied investigations, especially for small-volume sample analysis, on-site test and lab-on-a-chip assay (Li et al., 2020; Ozen et al., 2020). Using the precisely controlled fluids in small volume microfluidic channel, favorable, reliable, accurate, cost effective, and easy-to-use biosensors were developed (Dittrich et al., 2006; Whitesides 2006). For most of the microfluidic integrated biosensors, an interface covered by functional capture molecules plays a critical role for the recognition and capture of the targets in the microchannels (Foley et al., 2008). However, the binding efficiency of probes on the interface remains a major challenge to the development of microfluidic biosensors, because both the volume and the recognition time are limited in the microchannels (Lynn and Dandy 2007). The follow-on staggered herringbone microstructure that generates recirculation perpendicular to the primary flow direction is perhaps the best-known answer to improve the microfluidic recognition (Stroock et al., 2002; Williams et al., 2008). Unfortunately, such mixing approaches only increase the transport of the analytes in bulk solution, but the binding efficiency between analytes and probes on the interface still remains a bottleneck of microfluidics to overcome.

The two-phase interface where the electro migration, mass transfer, energy exchange and signal conversion happen, is the most critical for better biosensing performance (Helmke and Minerick 2006; Jones et al., 2014). Well-defined 3D self-assembled “framework nucleic acids” (FNAs) have drawn considerable research attention for the development of biosensor interface (Ge et al., 2018; Wang et al., 2019; Wiraja et al., 2019; Yang et al. 2018a, 2018b), with advantages such as simple synthesis, high yield, powerful structural rigidity and capacity of multiple functionalization with high addressability (Li et al., 2009; Liu et al., 2019; Song et al., 2020). Several reported biosensors demonstrated the high programmability of FNAs on the interface, leading to well defined orientation, density and configuration of the probes (Ge et al., 2020; Lin et al., 2015; Su et al., 2019).

Inspired by these works, a novel FNA, a tetrahedral DNA nanostructure (TDN), was designed and assembled onto the surface of the microchannel. Compared with macroscopic turbulence control on traditional micro-structured microfluidic surface, the novel TDN-engineered microfluidic interface successfully constructed a 3D binding space at nanoscale by raising DNA probes away from the surface. Thus, the reaction kinetics between the targets and the capture probes was dramatically improved.

As a proof of principle, we developed a novel multi-functional microfluidic biosensor for Escherichia coli (E. coli), which is one of the most common infections encountered in clinical practice and a common disease-causing agent affecting food safety (Hermann et al., 2019; Rajapaksha et al., 2019). The main challenges of conventional pathogen detection methods and drug sensitivity tests are analysis speed, sensitivity and requirement of intricate instruments and complex operation (Bush et al., 2011; Shin et al., 2019; Xu et al., 2016). As is well known, microfluidic assay is a very promising solution for rapid and sensitive pathogen analysis (Li et al., 2019; Liu et al., 2017; Qu et al., 2017). In the work, we successfully developed an integrated “one-stop” microfluidic biosensor, adapting the joint merits of microfluidic system and FNAs for pathogens monitoring. In this platform, each of its microchannels comprises an upstream herringbone channel for bacterial capture and a downstream microcavity structure for bacterial trapping, culture and AST. Finally, this compact microfluidic device with the built-in rigid FNA-aptamer probes and microcavities achieved ultrasensitive bacterial detection, fast and reliable AST analysis. Our work complements particularly the neglected molecular recognition between “dynamic analytes” and “static capture substrates”, other than most of the traditional microfluidic assays.