Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods.

We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ~56 nm and diameter ~12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

Highly efficient inhibition of human immunodeficiency virus type 1 reverse transcriptase by aptamers functionalized gold nanoparticles.

We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45-Au NPs shows inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity (40.2%).

Molecularly imprinted aptamers of gold nanoparticles for the enzymatic inhibition and detection of thrombin.

We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA(15)) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA(29)) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA(15)/TBA(29)-Tn-Au NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA(15)/TBA(29)-Tn-Au NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA(15)/TBA(29)-Tn-Au NPs, leading to their stronger binding strength with thrombin. MP-TBA(15)/TBA(29)-T(15)-Au NPs (ca. 42 TBA(15) and 42 TBA(29) molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10(-11) M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA(15)/TBA(29)-T(15)-Au NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA(15)/TBA(29)-T(15)-Au NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA(15)-T(15)-Au NPs and MP-TBA(29)-T(15)-Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins.

Colorimetric detection of platelet-derived growth factors through competitive interactions between proteins and functional gold nanoparticles.

We have developed a colorimetric assay-using aptamer modified 13-nm gold nanoparticles (Apt-Au NPs) and fibrinogen adsorbed Au NPs (Fib-Au NPs, 56nm)-for the highly selective and sensitive detection of platelet-derived growth factors (PDGF). Apt-Au NPs and Fib-Au NPs act as recognition and reporting units, respectively. PDGF-binding-aptamer (Apt(PDGF)) and 29-base-long thrombin-binding-aptamer (Apt(thr29)) are conjugated with Au NPs to prepare functional Apt-Au NPs (Apt(PDGF)/Apt(thr29)-Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacts with Fib-Au NPs in solutions to catalyze the formation of insoluble fibrillar fibrin-Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. The activity of thrombin is suppressed once it interacts with the Apt(PDGF)/Apt(thr29)-Au NPs. The suppression decreases due to steric effects through the specific interaction of PDGF with Apt(PDGF), occurring on the surfaces of Apt(PDGF)/Apt(thr29)-Au NPs. Under optimal conditions [Apt(PDGF)/Apt(thr29)-Au NPs (25pM), thrombin (400pM) and Fib-Au NPs (30pM)], the Apt(PDGF)/Apt(thr29)-Au NPs/Fib-Au NPs probe responds linearly to PDGF over the concentration range of 0.5-20nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio=3) for each of the three PDGF isoforms is 0.3nM in the presence of bovine serum albumin at 100µM. When using the Apt(PDGF)/Apt(thr29)-Au NPs as selectors for the enrichment of PDGF and for the removal of interferences from cell media, the LOD for PDGF provided by this probe is 35pM. The present probe reveals that the concentration of PDGF in the three cell media is 230 (±20)pM, showing its advantages of simplicity, sensitivity, and specificity.

Highly efficient control of thrombin activity by multivalent nanoparticles.

We have demonstrated that the incorporation of sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer (TBA)-conjugated gold nanoparticles (TBA-AuNPs) enables highly effective inhibition of thrombin activity toward fibrinogen. AuNP bioconjugates (TBA(15)/TBA(29)/sulf-Gal-AuNPs) were prepared from 13 nm AuNPs, 15-mer thrombin-binding aptamer (TBA(15)), 29-mer thrombin-binding aptamer (TBA(29)), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per AuNP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA(15)/TBA(29)/sulf-Gal-AuNPs (with 15 TBA(15) and 15 TBA(29) molecules per AuNP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (K(d)=3.4×10(-12) M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the "normal" value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA(15) alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequence (cTBA)-modified AuNPs (cTBA(15)/cTBA(29)-AuNPs) at a rate that was 20 times faster than that of free cTBA(15)/cTBA(29). Thus, easily prepared, low-cost, multivalent AuNPs show great potential for biomedical control of blood clotting.

Using self-assembled aptamers and fibrinogen-conjugated gold nanoparticles to detect DNA based on controlled thrombin activity.

We have developed a colorimetric probe, based on the aggregation of gold nanoparticles (Au NPs), for the detection of DNA and for the analysis of single-nucleotide polymorphism (SNP); this probe functions through the modulation of the activity of thrombin (Thr) in the presence of bivalent thrombin-binding aptamers (TBAs). The bivalent TBAs were formed from TBA(27') (comprising a 27-base sequence providing TBA(27) functionality, a T(5) linker, and an 11-base sequence for hybridization) and TBA(15') (comprising a 15-base sequence providing TBA(15) functionality, a T(5) linker, and a 12-base sequence for hybridization) through their hybridization with perfectly matched DNA (DNA(pm)). The bivalent TBAs interacted specifically with thrombin, suppressing its activity toward fibrinogen-modified Au NPs (Fib-Au NPs). The potency of the inhibitory effect of TBA(15')-TBA(27')/DNA(pm) toward thrombin - and, thus, the degree of aggregation of the Fib-Au NPs - was highly dependent on the concentration of DNA(pm). Under the optimal conditions (50 pM thrombin, 2 nM TBA(15'), 2 nM TBA(27'), and 38 pM Fib-Au NPs), the linear relationship of the response of the probe toward DNA(pm) extended from 0.1 to 2 nM, with a correlation coefficient of 0.97. The limit of detection (LOD) for DNA(pm) was 20 pM, based on a signal-to-noise ratio of 3. We also applied a corresponding TBA(15″)-TBA(27″)/Thr/Fib-Au NP probe to the detection of the SNP of the Arg249Ser unit in the TP53 gene, with an LOD of 32 pM. Relative to conventional molecular beacon-based and crosslinking aggregation-based Au NP probes, our new approach offers higher sensitivity and higher selectivity toward DNA.

Protein A-conjugated luminescent gold nanodots as a label-free assay for immunoglobulin G in plasma.

We have employed protein A-modified gold nanodots (PA-Au NDs) as a luminescence sensor for the detection of human immunoglobulin G (hIgG) in homogeneous solutions. The luminescent PA-Au NDs were prepared simply by mixing protein A with the luminescent Au NDs (average diameter: ca. 1.8 nm). The specific interactions that occur between protein A and hIgG allowed us to use the PA-Au NDs to detect hIgG selectively. Under optimal conditions [10 nM PA-Au NDs (two protein A molecules per Au ND), 5.0 mM phosphate buffer solution, pH 7.4], the PA-Au ND probe detected hIgG with high sensitivity (limit of detection = 10 nM) and remarkable selectivity (>50-fold) over other proteins. In an assay that took advantage of the competition between protein G and the PA-Au NDs for IgG, we detected protein G at concentrations as low as 85 nM. This PA-Au ND probe allowed determination of the hIgG concentration in plasma samples without any need for sample pretreatment. Our results exhibited a good linear correlation (R(2)=0.97) with those obtained using an enzyme-linked immunosorbent assay. Our simple, sensitive, and selective approach appears to hold practical potential for use in the clinical diagnosis of immune diseases associated with changes in hIgG levels.

Photoassisted synthesis of luminescent mannose-Au nanodots for the detection of thyroglobulin in serum.

We have employed mannose-modified gold nanodots (Man-Au NDs) as a luminescence sensor for the detection of the thyroid-cancer marker thyroglobulin (Tg) in homogeneous solutions. The luminescent Man-Au NDs are prepared through the reaction of 2.9 nm-diameter gold nanoparticles (Au NPs) with 11-mercapto-3,6,9-trioxaundecyl-alpha-D-mannopyranoside (Man-RSH) under the irradiation of a light-emitting diode (LED). We have found that the irradiation enhances the quantum yield (approximately 11%), alters the emission wavelength and lifetimes, and shortens the preparation time. A luminescence assay has been developed for Tg based on the competition between Tg and Man-Au NDs for the interaction with the concanavalin A (Con A). Because luminescence quenching of the Man-Au NDs by Con A is inhibited by Tg selectivity, we have obtained a highly sensitive and selective assay for Tg.

Colorimetric assay for lead ions based on the leaching of gold nanoparticles.

A colorimetric, label-free, and nonaggregation-based gold nanoparticles (Au NPs) probe has been developed for the detection of Pb(2+) in aqueous solution, based on the fact that Pb(2+) ions accelerate the leaching rate of Au NPs by thiosulfate (S(2)O(3)(2-)) and 2-mercaptoethanol (2-ME). Au NPs reacted with S(2)O(3)(2-) ions in solution to form Au(S(2)O(3))(2)(3-) complexes on the Au NP surfaces, leading to slight decreases in their surface plasmon resonance (SPR) absorption. Surface-assisted laser desorption/ionization time-of-flight ionization mass spectrometry (SALDI-TOF MS) data reveals the formation of Pb-Au alloys on the surfaces of the Au NPs in the presence of Pb(2+) ions and 2-ME. The formation of Pb-Au alloys accelerated the Au NPs rapidly dissolved into solution, leading to dramatic decreases in the SPR absorption. The 2-ME/S(2)O(3)(2-)-Au NP probe is highly sensitive (LOD = 0.5 nM) and selective (by at least 1000-fold over other metal ions) toward Pb(2+) ions, with a linear detection range (2.5 nM-10 muM) over nearly 4 orders of magnitude. The cost-effective probe allows rapid and simple determination of the concentrations of Pb(2+) ions in environmental samples (Montana soil and river), with results showing its great practicality for the detection of lead in real samples.

Gold nanodot-based luminescent sensor for the detection of hydrogen peroxide and glucose.

We unveil a new luminescent assay using 11-mercaptoundecanoic acid-bound Au nanodots (11-MUA-Au NDs) for the highly selective and sensitive detection of hydrogen peroxide and glucose, based on luminescence quenching.

Synthesis of fluorescent carbohydrate-protected Au nanodots for detection of Concanavalin A and Escherichia coli.

This study describes a novel, simple, and convenient method for the preparation of water-soluble biofunctional Au nanodots (Au NDs) for the detection of Concanavalin A (Con A) and Escherichia coli (E. coli). First, 2.9 nm Au nanoparticles (Au NPs) were prepared through reduction of HAuCl(4).3H(2)O with tetrakis(hydroxymethyl)phosphonium chloride (THPC), which acts as both a reducing and capping agent. Addition of 11-mercapto-3,6,9-trioxaundecyl-alpha-D-mannopyranoside (Man-SH) onto the surfaces of the as-prepared Au NPs yielded the fluorescent mannose-protected Au nanodots (Man-Au NDs) with the size and quantum yield (QY) of 1.8 (+/-0.3) nm and 8.6%, respectively. This QY is higher than those of the best currently available water-soluble, alkanethiol-protected Au nanoclusters. Our fluorescent Man-Au NDs are easily purified and by multivalent interactions are capable of sensing, under optimal conditions, Con A with high sensitivity (LOD = 75 pM) and remarkable selectivity over other proteins and lectins. To the best of our knowledge, this approach provided the lowest LOD value for Con A when compared to the other nanomaterials-based detecting method. Furthermore, we have also developed a new method for fluorescence detection of E. coli using these water-soluble Man-Au NDs. Incubation with E. coli revealed that the Man-Au NDs bind to the bacteria, yielding brightly fluorescent cell clusters. The relationship between the fluorescence signal and the E. coli concentration was linear from 1.00 x 10(6) to 5.00 x 10(7) cells/mL (R(2) = 0.96), with the LOD of E. coli being 7.20 x 10(5) cells/mL.