Mechanism Studies on NanoPCR and Applications of Gold Nanoparticles in Genetic Analysis

Recently, the applications of nanomaterial-assisted polymerase chain reaction (nanoPCR) have received considerable attention. Several potential mechanisms have been proposed, but mainly according to the results of PCR assays under specific conditions and lacking direct and general evidence. The mechanism of nanoPCR has not been elucidated yet. Here, taking gold nanoparticles (AuNPs) as an example, we report the three general effects of AuNPs: (1) AuNPs adsorb polymerase and modulate the amount of active polymerase in PCR, which was directly demonstrated by a simple and straightforward colorimetric assay and the dynamic light scattering measurements. (2) AuNPs adsorb primers and decrease the melting temperatures (<i>T</i>_m) of the duplexes formed with perfectly matched and mismatched primers and increase the <i>T</i>_m difference between them. (3) AuNPs adsorb PCR products and facilitate the dissociation of them in the denaturing step. All these effects were confirmed by addition of a rationally selected surface adsorbent, bovine thrombin, to highly efficiently modulate the surface adsorption of PCR components. These findings suggested that AuNPs should have multiple effects on PCR: (1) to regulate PCR in a case-by-case way via modulating the amount of active polymerase in PCR; (2) to improve PCR specificity in the annealing step via increasing the <i>T</i>_m difference between the perfectly matched and mismatched primers; (3) to improve PCR efficiency via speeding up the dissociation of the PCR products in the denaturing step. Taken together, we proposed the mechanism of nanoPCR is that the surface interaction of PCR components (polymerase, primers, and products) with AuNPs regulates nanoPCR. We further demonstrated that the applications of these findings improve the PCR of the amelogenin genes and Hepatitis B virus gene for genetic analysis. These findings could also provide helpful insight for the applications of other nanomaterials in nanoPCR

Label-Free Fluorescent Detection of Ions, Proteins, and Small Molecules Using Structure-Switching Aptamers, SYBR Gold, and Exonuclease I

We have demonstrated a label-free sensing strategy employing structure-switching aptamers (SSAs), SYBR Gold, and exonuclease I to detect a broad range of targets including inorganic ions, proteins, and small molecules. This nearly universal biosensor approach is based on the observation that SSAs at binding state with their targets, which fold into secondary structures such as quadruplex structure or Y shape structure, show more resistance to nuclease digestion than SSAs at unfolded states. The amount of aptamer left after nuclease reaction is proportional to the concentrations of the targets and in turn is proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates (dNMPs). Fluorescent assays employing this mechanism for the detection of potassium ion (K⁺) are sensitive, selective, and convenient. Twenty μM K⁺ is readily detected even at the presence of a 500-fold excess of Na⁺. Likewise, we have generalized the approach to the specific and convenient detection of proteins (thrombin) and small molecules (cocaine). The assays were then validated by detecting K⁺, cocaine, and thrombin in urine and serum or cutting and masking adulterants with good agreements with the true values. Compared to other reported approaches, most limited to G-quadruplex structures, the demonstrated method has less structure requirements of both the SSAs and their complexes with targets, therefore rending its wilder applications for various targets. The detection scheme could be easily modified and extended to detection platforms to further improve the detection sensitivity or for other applications as well as being useful in high-throughput and paralleled analysis of multiple targets

Self-Assembled DNA Monolayer Buffered Dynamic Ranges of Mercuric Electrochemical Sensor

Sensors with wide dynamic ranges (DRs) are typically constructed by utilizing a set of ligands with varied affinities for the same target. We report here a novel buffer self-assembled monolayer (BSAM) strategy, to fabricate sensors with extraordinarily broad DRs using a single recognition ligand. We demonstrated the concept of BSAM by constructing the electrochemical mercuric sensors with different surface probe densities (SPD) on a gold electrode. These sensors are based on the coordination of Hg²⁺ with a pair of thymine (T) formed between the two proximate poly­(T) oligonucleotides on the electrode surface and Hg²⁺ binding induced DNA strand displacement of ferrocene tagged poly­(A). There are three types of T–Hg²⁺–T coordination: those formed between (a) two poly­(T) strands where none are hybridized with poly­(A) strands, thus contributing zero effect on releasing the signaling probe, (b) poly­(A)/poly­(T) hybridized and nonhybridized poly­(T) strands, resulting in the release of a signaling probe from the surface; and (c) two poly­(A)/poly­(T) hybridized strands, causing the release of two signaling probes from the surface. The DRs from 10 pM to 0.1 mM at varied SPDs were observed, attributing to the tunable Hg²⁺ storage capability of the poly­(T) SAM formed on the surface due to the coordination mechanism of (a) and (b). The DR was able to be further extended to 1 mM by using the longer poly­(T) strands. The ready-to-use sensor exhibited great selectivity against the common interferential metal ions. As demonstrated, the BSAM strategy is a facile way to fabricate sensors with tunable and wide DRs

Rapid, Surfactant-Free, and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA under Physiological pH and Its Application in Molecular Beacon-Based Biosensor

The controlled attachment of thiolated DNA to gold nanoparticles (AuNPs) dictates many applications. This is typically achieved by either “aging-salting” processes or low-pH method, where either Na⁺ or H⁺ is used to minimize charge repulsion and facilitate attachment of thiolated DNA onto AuNPs. However, the “aging-salting” process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants are needed to precoat and thereby enhance the stability of AuNPs. The low-pH method can disrupt the structural integrity of DNAs. We report here an oligoethylene glycol (OEG) spacer-assisted method that enables quantitative and instantaneous attachment at physiological pH without the need for surfactants. The method is based on our finding that an uncharged OEG spacer as short as six EG units can effectively shield against repulsion between AuNPs and DNAs, substantially enhancing both the adsorption kinetics and thermodynamics of thiolated DNAs. We applied this to thiolated DNAs of various lengths and thiol modification positions and to large AuNPs. Importantly, our method also allows for the direct immobilization of thiolated molecular beacons (MB), and avoids particle aggregation due to intermolecular hydrogen bonding. The prepared MB-AuNPs were successfully used for the fluorescent detection of target DNA at nanomolar concentrations. The OEG spacer appears to offer a highly effective parameter for tuning DNA adsorption kinetics and thermodynamics besides pH and salt, providing a novel means for highly controllable and versatile functionalization of AuNPs

Amplified Single Base-Pair Mismatch Detection via Aggregation of Exonuclease-Sheared Gold Nanoparticles

Single nucleotide polymorphism (SNP) detection is important for early diagnosis, clinical prognostics, and disease prevention, and a rapid and sensitive low-cost SNP detection assay would be valuable for resource-limited clinical settings. We present a simple platform that enables sensitive, naked-eye detection of SNPs with minimal reagent and equipment requirements at room temperature within 15 min. SNP detection is performed in a single tube with one set of DNA probe-modified gold nanoparticles (AuNPs), a single exonuclease (Exo III), and the target in question. Exo III’s apurinic endonucleolytic activity differentially processes hybrid duplexes between the AuNP-bound probe and DNA targets that are perfectly matched or contain a single-base mismatch. For perfectly matched targets, Exo III’s exonuclease activity facilitates a process of target recycling that rapidly shears DNA probes from the particles, generating an AuNP aggregation-induced color change, whereas no such change occurs for mismatched targets. This color change is easily observed with as little as 2 nM of target, 100-fold lower than the target concentration required for reliable naked eye observation with unmodified AuNPs in well-optimized reaction conditions. We further demonstrate that this system can effectively discriminate a range of different mismatches

A Label-Free Aptamer-Fluorophore Assembly for Rapid and Specific Detection of Cocaine in Biofluids

We report a rapid and specific aptamer-based method for one-step cocaine detection with minimal reagent requirements. The feasibility of aptamer-based detection has been demonstrated with sensors that operate via target-induced conformational change mechanisms, but these have generally exhibited limited target sensitivity. We have discovered that the cocaine-binding aptamer MNS-4.1 can also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naph­thyridine (ATMND) and thereby quench its fluorescence. We subsequently introduced sequence changes into MNS-4.1 to engineer a new cocaine-binding aptamer (38-GC) that exhibits higher affinity to both ligands, with reduced background signal and increased signal gain. Using this aptamer, we have developed a new sensor platform that relies on the cocaine-mediated displacement of ATMND from 38-GC as a result of competitive binding. We demonstrate that our sensor can detect cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than existing assays based on target-induced conformational change. More importantly, our assay achieves successful cocaine detection in body fluids, with a limit of detection of 10.4, 18.4, and 36 μM in undiluted saliva, urine, and serum samples, respectively

Highly Sensitive and Selective Chip-Based Fluorescent Sensor for Mercuric Ion: Development and Comparison of Turn-On and Turn-Off Systems

Miniaturization is currently an important trend in environmental and food monitoring because it holds great promise for on-site monitoring and detection. We report here two ready-to-use chip-based fluorescent sensors, compatible with microarray technology for reagentless, one-step, fast, highly sensitive and selective detection of the mercuric ion (Hg²⁺) in the turn-on and turn-off operation modes. Both operation modes are based on the highly selective T–Hg²⁺–T coordination between two neighboring polythymine (T) strands at a high probe density and its induced displacement of the complementary polyadenine strand labeled with either fluorophore or quencher, which enables the turn-off and turn-on detection of Hg²⁺, respectively. The turn-off sensor is slightly more sensitive than the turn-on sensor, and their detection limits are 3.6 and 8.6 nM, respectively, which are both lower than the U.S. Environmental Protection Agency limit of [Hg²⁺] for drinkable water (10 nM, 2 ppb). Compared to the turn-off sensor with the dynamic Hg²⁺ detection range from 3.6 nM to 10 μM (<i>R</i>² = 0.99), the turn-on sensor has a broader dynamic Hg²⁺ detection range, from 8.6 nM to 100 μM (<i>R</i>² = 0.996). Both sensors exhibited superior selectivity over other reported sensors using thymine-rich probes for Hg²⁺ detection over other common metal ions. In addition, the practical application of the chip-based sensors was demonstrated by detecting spiked Hg²⁺ in drinking water and fresh milk. The sensor has great potential for on-site practical applications due to its operational convenience, simplicity, speed, and portability

Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000–100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms