Yingfu Li

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.

Biologically Inspired Synthetic Enzymes Made from DNA

In cells, DNA typically consists of two antiparallel strands arranged in a double-helical structure, which is central to its fundamental role in storing and transmitting genetic information. In laboratories, however, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures, including some that have been shown to catalyze chemical transformations. These catalytic DNA molecules are commonly referred to as DNAzymes, or deoxyribozymes. Thus far, DNAzymes have not been found in cells, but hundreds of structural and functional variations have been created in the laboratory. This alternative catalytic platform has piqued the curiosity of many researchers, including those who seek to exploit them in potential applications ranging from analytical tools to therapeutic agents. In this review, we explore the unconventional role of DNA as a biologically inspired synthetic enzyme.

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

Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences.

We report a group of new DNA enzymes that possess a synchronized RNA-cleavage/fluorescence-signaling ability and exhibit wide-ranging metal-ion and pH dependences. These DNA catalysts were derived from a random-sequence DNA pool in a two-stage process: (1) establishment of a catalytic DNA population through repetitive rounds of in vitro selection at pH 4.0, and (2) sequence-diversification and catalytic-activity optimization through five parallel paths of in vitro evolution conducted at pH 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The deoxyribozymes were evolved to cleave the phosphodiester bond of a single ribonucleotide embedded in DNA and flanked immediately by two deoxyribonucleotides modified with a fluorophore and a quencher, respectively--a setting that synchronizes catalysis with fluorescence signaling. The most dominant catalyst from each pool was examined for metal-ion specificity, catalytic efficiency, pH dependence, and fluorescence-signaling capability. Individual catalysts have different metal-ion requirements and can generate as much as a 12-fold fluorescence enhancement upon RNA cleavage. Most of the DNA enzymes have a pH optimum coinciding with the selection pH and exhibit a rate constant approximating 1 min(-)(1) under optimal reaction conditions. The demonstration of DNA enzymes that are functional under extremely high acidity (such as pH 3 and 4) indicates that DNA has the ability to perform efficient catalysis even under harsh reaction conditions. The isolation of many new signaling DNA enzymes with broad pH optima and metal-ion specificities should facilitate the development of diverse deoxyribozyme-based biosensors.

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.

Colorimetric Sensing by Using Allosteric-DNAzyme-Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe†

Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

Microgel-based inks for paper-supported biosensing applications.

As a first step for the development of biosensing inks for inexpensive paper-based biodetection, we prepared paper strips printed with carboxylic poly( N-isopropylacrylamide) microgels that were modified either with an antibody or with a DNA aptamer. We found that the antibody and the DNA aptamer retained their recognition capabilities when coupled to microgel. The printed microgel remains stationary during chromatographic elution while the microgel-supported molecular recognition elements are accessible to their intended targets present in the elution solution. Our work indicates that microgels, large enough to isolate the biosensors from the paper surface, are sufficiently hydrophilic to be wetted during chromatographic elution, exposing the gel-supported affinity probes to their targets.

Recent Progress in Nucleic Acid Aptamer-Based Biosensors and Bioassays

As the key constituents of the genetic code, the importance of nucleic acids to life has long been appreciated. Despite being composed of only four structurally similar nucleotides, single-stranded nucleic acids, as in single-stranded DNAs and RNAs, can fold into distinct three-dimensional shapes due to specific intramolecular interactions and carry out functions beyond serving as templates for protein synthesis. These functional nucleic acids (FNAs) can catalyze chemical reactions, regulate gene expression, and recognize target molecules. Aptamers, whose name is derived from the Latin word aptus meaning “to fit”, are oligonucleotides that can bind their target ligands with high affinity and specificity. Since aptamers exist in nature but can also be artificially isolated from pools of random nucleic acids through a process called in vitro selection, they can potentially bind a diverse array of compounds. In this review, we will discuss the research that is being done to develop aptamers against various biomolecules, the progress in engineering biosensors by coupling aptamers to signal transducers, and the prospect of employing these sensors for a range of chemical and biological applications. Advances in aptamer technology emphasizes that nucleic acids are not only the fundamental molecules of life, they can also serve as research tools to enhance our understanding of life. The possibility of using aptamer-based tools in drug discovery and the identification of infectious agents can ultimately augment our quality of life.