Jiuxing Li

Label-Free Surface-Enhanced Raman Spectroscopy Detection of DNA with Single-Base Sensitivity

Direct, label-free detection of unmodified DNA is a great challenge for DNA analyses. Surface-enhanced Raman spectroscopy (SERS) is a promising tool for DNA analyses by providing intrinsic chemical information with a high sensitivity. To address the irreproducibility in SERS analysis that hampers reliable DNA detection, we used iodide-modified Ag nanoparticles to obtain highly reproducible SERS signals of single- and double-strand DNA in aqueous solutions close to physiological conditions. The phosphate backbone signal was used as an internal standard to calibrate the absolute signal of each base for a more reliable determination of the DNA structure, which has not been achieved before. Clear identification of DNA with single-base sensitivity and the observation of a hybridization event have been demonstrated.

Translating Molecular Recognition into a Pressure Signal to enable Rapid, Sensitive, and Portable Biomedical Analysis

Herein, we demonstrate that a very familiar, yet underutilized, physical parameter—gas pressure—can serve as signal readout for highly sensitive bioanalysis. Integration of a catalyzed gas-generation reaction with a molecular recognition component leads to significant pressure changes, which can be measured with high sensitivity using a low-cost and portable pressure meter. This new signaling strategy opens up a new way for simple, portable, yet highly sensitive biomedical analysis in a variety of settings.

Design and Synthesis of Target-Responsive Aptamer-Cross-linked Hydrogel for Visual Quantitative Detection of Ochratoxin A

A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core-shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.

Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2

Background Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. Methodology/Principal Findings Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with Kd values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. Conclusion/Significance In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

Integration of target responsive hydrogel with cascaded enzymatic reactions and microfluidic paper-based analytic devices (µPADs) for point-of-care testing (POCT).

Paper based microfluidics (µPADs) with advantages of portability, low cost, and ease of use have attracted extensive attention. Here we describe a novel method that integrates glucoamylase-trapped aptamer-crosslinked hydrogel for molecular recognition with cascaded enzymatic reactions for signal amplification and a µPAD for portable readout. Upon target introduction, the hydrogel decomposes to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose. With a simple folding of the µPAD, the sample solution containing glucose product wicks and diffuses in parallel to each test-zone to carry out homogeneous assays, where glucose is used to produce I2 for brown color visualization through multiple enzymatic and chemical cascade reactions. Through color gradient changes based on different concentrations of the target, a semiquantitative assay is achieved by the naked eye, and quantitation can be obtained by handheld devices. Detection of cocaine in buffer and urine was performed to demonstrate the utility of the hydrogel-µPAD system. More importantly, the hydrogel-µPAD system can be extended to the detection of various targets by incorporating the corresponding aptamer into the hydrogel. The hydrogel-µPAD system reported here provides a new platform for portable, disposable and visual detection of a wide range of targets.

Surface-Enhanced Raman Scattering Active Plasmonic Nanoparticles with Ultrasmall Interior Nanogap for Multiplex Quantitative Detection and Cancer Cell Imaging

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

Synergetic Approach for Simple and Rapid Conjugation of Gold Nanoparticles with Oligonucleotides

Attaching thiolated DNA on gold nanoparticles (AuNPs) has been extremely important in nanobiotechnology because DNA-AuNPs combine the programmability and molecular recognition properties of the biopolymers with the optical, thermal, and catalytic properties of the inorganic nanomaterials. However, current standard protocols to attach thiolated DNA on AuNPs involve time-consuming, tedious steps and do not perform well for large AuNPs, thereby greatly restricting applications of DNA-AuNPs. Here we demonstrate a rapid and facile strategy to attach thiolated DNA on AuNPs based on the excellent stabilization effect of mPEG-SH on AuNPs. AuNPs are first protected by mPEG-SH in the presence of Tween 20, which results in excellent stability of AuNPs in high ionic strength environments and extreme pHs. A high concentration of NaCl can be applied to the mixture of DNA and AuNP directly, allowing highly efficient DNA attachment to the AuNP surface by minimizing electrostatic repulsion. The entire DNA loading process can be completed in 1.5 h with only a few simple steps. DNA-loaded AuNPs are stable for more than 2 weeks at room temperature, and they can precisely hybridize with the complementary sequence, which was applied to prepare core-satellite nanostructures. Moreover, cytotoxicity assay confirmed that the DNA-AuNPs synthesized by this method exhibit lower cytotoxicity than those prepared by current standard methods. The proposed method provides a new way to stabilize AuNPs for rapid and facile loading thiolated DNA on AuNPs and will find wide applications in many areas requiring DNA-AuNPs, including diagnosis, therapy, and imaging.

Simple and Rapid Functionalization of Gold Nanorods with Oligonucleotides Using an mPEG-SH/Tween 20-Assisted Approach

DNA conjugated gold nanorods (AuNRs) are widely applied for nanostructure assembly, gene therapy, biosensing, and drug delivery. However, it is still a great challenge to attach thiolated DNA on AuNRs, because the positively charged AuNRs readily aggregate in the presence of negatively charged DNA. This article reports an mPEG-SH/Tween 20-assisted method to load thiolated DNA on AuNRs in 1 h. Tween 20 and mPEG-SH are used to synergistically displace CTAB on the surface of AuNRs by repeated centrifugation and resuspension, and thiolated DNA are attached to AuNRs in the presence of 1 M NaCl, 100 mM MgCl2, or 100 mM citrate. AuNRs with different sizes and aspect ratios can be functionalized with DNA by this method. The number of DNA loaded on each AuNR can be easily controlled by the concentrations of mPEG-SH and Tween 20 or the ratio between DNA and AuNR. Functionalized AuNRs were used for nanoparticle assembly and cancer cell imaging to confirm that DNA anchored on the surface of AuNRs retains its hybridization and molecular recognition capability. The new method is easy, rapid, and robust for the preparation of DNA functionalized AuNRs for a variety of applications such as cancer therapy, drug delivery, self-assembly, and imaging.

Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-Friendly, Quantitative Point-of-Care Testing

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.

DNA‐Mediated Morphological Control of Silver Nanoparticles

It is demonstrated that DNA can be used to control the synthesis of silver nanoplates with different morphologies using spherical silver seeds. UV-vis spectroscopy, transmission electron microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy are used to characterize the synthesized nanoparticles. Silver nanoprisms are encoded by poly C and poly G, while silver flower bouquets and silver nanodiscs are synthesized using poly A and poly T, respectively. The length of DNA is found to have little effect on the morphology of silver nanoparticles. Moreover, the synthesized silver nanoplates are found to have high surface enhanced Raman scattering enhancement ability, good antibacterial activity, and good biocompatibility. These discoveries will broaden the application of DNA in nanoscience and will provide a new platform to investigate the interaction between DNA sequences and silver nanoparticles.