Weian Zhao

Design of gold nanoparticle-based colorimetric biosensing assays.

Gold nanoparticle (AuNP)‐based colorimetric biosensing assays have recently attracted considerable attention in diagnostic applications due to their simplicity and versatility. This Minireview summarizes recent advances in this field and attempts to provide general guidance on how to design such assays. The key to the AuNP‐based colorimetric sensing platform is the control of colloidal AuNP dispersion and aggregation stages by using biological processes (or analytes) of interest. The ability to balance interparticle attractive and repulsive forces, which determine whether AuNPs are stabilized or aggregated and, consequently, the color of the solution, is central in the design of such systems. AuNP aggregation in these assays can be induced by an “interparticle‐crosslinking” mechanism in which the enthalpic benefits of interparticle bonding formation overcome interparticle repulsive forces. Alternatively, AuNP aggregation can be guided by the controlled loss of colloidal stability in a “noncrosslinking‐aggregation” mechanism. In this case, as a consequence of changes in surface properties, the van der Waals attractive forces overcome interparticle repulsive forces. Using representative examples we illustrate the general strategies that are commonly used to control AuNP aggregation and dispersion in AuNP‐based colorimetric assays. Understanding the factors that play important roles in such systems will not only provide guidance in designing AuNP‐based colorimetric assays, but also facilitate research that exploits these principles in such areas as nanoassembly, biosciences and colloid and polymer sciences.

Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.

DNA Aptamer Folding on Gold Nanoparticles: From Colloid Chemistry to Biosensors

We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.

Simple and Rapid Colorimetric Biosensors Based on DNA Aptamer and Noncrosslinking Gold Nanoparticle Aggregation

Recently, gold nanoparticles (AuNPs) have emerged as novel colorimetric reporters for the detection of various substances including DNA, metal ions, and proteins. The advantages of using AuNPs include: 1) their simplicity, 2) the fact that no complicated and expensive analytical instruments are needed, and 3) the extremely high extinction coefficients ( 1000 times larger than those of organic dyes) and the strongly distance-, shape-, and size-dependent optical properties of AuNPs, which allow AuNP-based colorimetric detection to have comparable sensitivity and selectivity to conventional fluorescent detection. AuNP’s use as a colorimetric reporter relies on its unique surface plasmon resonance (SPR): the dispersed AuNP solution is red whereas the aggregated AuNP solution appears purple (or blue), a phenomenon that can be well explained by the Mie theory. Based on this principle, two general types of colorimetric assays (referred to as type I and type II in this report) have been developed. In type I assays, the color of the AuNP solution changes from red (dispersed particles) to purple (aggregates), in type II assays, the color changes from purple (aggregates) to red (dispersed particles). Mirkin and co-workers pioneered the type I assay in which AuNP was used for the detection of DNA. In their study, AuNPs that were modified with two different oligonucleotides aggregated upon the addition of the complementary DNA target, which acted as a crosslinker to result in a color change from red to purple. Liu and Lu reported a type II assay for the detection of lead ions in which the aggregated AuNPs, crosslinked by cleavable DNA enzymes, were dissociated into dispersed AuNPs in the presence of Pb . More recently, Liu and Lu have extended this concept for the detection of small organic compounds (such as ATP) by using AuNPs crosslinked by DNA aptamers. Aptamers are single-stranded (ss) DNA or RNA molecules created by in vitro selection for binding to a chosen target with high affinity and specificity. In the aptamer-based assay designed by Liu and Lu, oligonucleotide-modified AuNPs were first crosslinked by a DNA aptamer sequence to form aggregates. Upon the addition of a desirable target, the aptamer underwent a structural switch that caused the dissociation of the AuNP aggregates; this was accompanied by the purple-to-red color change. The marriage of AuNP and aptamers in these studies allows the AuNP-based assay to be generic, in principle, for any analyte for which an appropriate aptamer is available. We present here a simple and rapid colorimetric assay that exploits structure-switching DNA aptamers and the phenomenon of salt-induced, noncrosslinking AuNP aggregation. Conceptually, as shown in Figure 1A, a structure-switching DNA

Lab on paper

Lab-on-a-chip (LOC) devices, which are suited to portable point-of-care (POC) diagnostics and on-site detection, hold great promise for improving global health, and other applications.1–8 While their importance and utility are widely acknowledged and extensive research has been conducted in the laboratory on device manipulation and proof-ofconcept demonstration, there are few commercialized LOC products that are fabricated using clean-room based technologies

Bioinspired multivalent DNA network for capture and release of cells

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Engineered cell homing

One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors.

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.

Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.

Tracking mesenchymal stem cells with iron oxide nanoparticle loaded poly(lactide-co-glycolide) microparticles.

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4-3 μm) in MSCs enhances MR parameters such as the r(2) relaxivity (5-fold), residence time inside the cells (3-fold) and R(2) signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation.