In this paper, we present for the first time a single gold nanoparticle counter (SGNPC) in solution based on the photon bursting in a highly focused laser beam (less than 1 fL) due to the plasmon resonance scattering and Brownian motion of gold nanoparticles (GNPs). The photon burst intensity of single 36 nm GNPs is several tens to hundreds times stronger than that of quantum dots (QDs) and organic dyes. The relationship between the photon burst counts and GNPs concentration shows an excellent linearity. The linear range is over 4 orders of magnitude, and the detection limit of GNPs (36 nm) is 17 fM. On the basis of this single nanoparticle technique, we developed an ultrasensitive and highly selective detection platform for homogeneous immunoassay and DNA hybridization assays using GNPs as probes, which were 2-5 orders of magnitude more sensitive than current homogeneous methods. We used this technology to construct homogeneous sandwich immunoassays for cancer biomarkers, such as carcinoembryonic antigen (CEA) and alpha fetal protein (AFP), and aptamer recognition for thrombin. The detection limits are 130 fM for CEA, 714 fM for AFP and 2.72 pM for thrombin. Our method was successfully applied for direct determination of CEA, AFP and thrombin levels in sera from healthy subjects and cancer patients. In homogeneous DNA hybridization detection, we chose methylenetetrahydrofolate reductase (MTHFR) gene as a target. This assay successfully distinguished DNA sequences with single base mismatches, and the detection limits for the target were at 1 fM level.

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Synthesis of gold nanoparticles (GNPs).GNPs were synthesized by reduction of the HAuCl 4 solution with sodium citrate. 1 Briefly, 100 mL of 0.01% (w/v) of the HAuCl 4 solution was heated to boiling for 15 minutes, and then 1% (w/v) sodium citrate solution was added. The volume added depended on the requirement size of GNPs, and 1 mL of sodium citrate solution was needed for 36 nm GNPs. Heating continued for 15 minutes after the solution color remained unchanged. After cooling down to room temperature, the prepared GNPs stock solution was stored at 4 o C for further use. The concentration of GNPs was calculated according to the reference. 2 GNPs were further purified and concentrated by centrifugation (Beckman Coulter, USA) 2 times at 2,500 rpm for 30 min (washing buffer: 0.01 M PBS, pH 7.4) prior to further use. ELISA method (contrast experiments).To investigate the reliability of our method, the human sera were analyzed with ELISA method according to the operational manuals of ELISA kits. Briefly, 50 μL antigen or human serum samples were added to the wells of the ELISA plate, and incubated at 37 o C for

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DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single-silver-nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon-burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single-nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag-NP-oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon-burst counts. The SSNPC method was used to measure the change in the photon-burst counts. The relationship between the change of the photon-burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence-specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2-5 orders of magnitude more sensitive than current homogeneous methods.