Effect of Serum on Electrochemical Detection of Bioassays Having Ag Nanoparticle Labels.

The effect of serum on electrochemical detection of bioassays having silver nanoparticle (AgNP) detection labels was investigated. Both a model assay and an antigen-specific sandwich bioassay for the heart failure marker NT-proBNP were examined. In both cases, the AgNP labels were conjugated to a detection antibody. Electrochemical detection was carried out using a galvanic exchange/anodic stripping voltammetry method in which Au3+ exchanges with AgNP labels. The assays were carried out using a paper-based electrode platform. The bioassays were exposed to different serum conditions prior to and during detection. There are three important outcomes reported in this article. First, both the model- and antigen-specific assays could be formed in undiluted serum with no detectable interferences from the serum components. Second, to achieve the maximum possible electrochemical signal, the highest percentage of serum that can remain in an assay buffer during electrochemical detection is 0.25% when no washing is performed. The assay results are rendered inaccurate when 0.50% or more of serum is present. Third, the factors inhibiting galvanic exchange in serum probably relate to surface adsorption of biomolecules onto the AgNP labels, chelation of Au3+ by serum components, or both. The results reported here provide general guidance for using metal NP labels for electrochemical assays in biofluids.

Silver Nanocubes as Electrochemical Labels for Bioassays.

Here, we report on the use of 40 ± 4 nm silver nanocubes (AgNCs) as electrochemical labels in bioassays. The model metalloimmunoassay combines galvanic exchange (GE) and anodic stripping voltammetry (ASV). The results show that a lower limit of detection is achieved by simply changing the shape of the Ag label yielding improved GE with AgNCs when compared to GE with spherical silver nanoparticles (sAgNPs). Specifically, during GE between electrogenerated Au3+ and the Ag labels, a thin shell of Au forms on the surface of the NP. This shell is more porous when GE proceeds on AgNCs compared to sAgNPs, and therefore, more exchange occurs when using AgNCs. ASV results show that the Ag collection efficiency (AgCE%) is increased by up to ∼57% when using AgNCs. When the electrochemical system is fully optimized, the limit of detection is 0.1 pM AgNCs, which is an order of magnitude lower than that of sAgNP labels.

Dual-Shaped Silver Nanoparticle Labels for Electrochemical Detection of Bioassays.

In this paper we demonstrate the use of dual-shaped silver nanoparticles (AgNPs) as detection labels for electrochemical bioassays. The key finding is that by using AgNP labels having two different shapes simultaneously, the limit of detection (LOD) for the assays is lowered compared to using either of the two shapes separately. The two shapes were silver nanocubes (AgNCs) having edge lengths of 40 ± 4 nm and spherical AgNPs (sAgNPs) having diameters of 20 ± 3 nm. Two different bioassays were examined. In both cases the Ag labels were functionalized with antibodies. In the one assay, the labels are directly linked to a second antibody immobilized on magnetic beads. In the second assay, the antibodies on the AgNP labels and the antibodies on the magnetic beads are linked via a peptide. The peptide is N-terminal prohormone brain natriuretic peptide (NT-proBNP), which is a heart failure marker. The efficacy of the two electrochemical assays as a function of the ratio of the two labels was investigated using a galvanic exchange/anodic stripping voltammetry method. The key finding is that by optimizing the ratio of the two types of AgNP labels, it is possible to decrease the LOD of the assays without compromising the dynamic range compared to using either of the two labels independently. This made it possible to achieve the clinically relevant range for NT-proBNP analysis used by physicians for heart failure risk stratification.

Electrochemical Detection of NT-proBNP Using a Metalloimmunoassay on a Paper Electrode Platform.

In this paper, we demonstrate an electrochemical method for detection of the heart failure biomarker, N-terminal prohormone brain natriuretic peptide (NT-proBNP). The approach is based on a paper electrode assembly and a metalloimmunoassay; it is intended for eventual integration into a home-use sensor. Sensing of NT-proBNP relies on the formation of a sandwich immunoassay and electrochemical quantification of silver nanoparticle (AgNP) labels attached to the detection antibodies (Abs). There are four important outcomes reported in this article. First, compared to physisorption of the detection Abs on the AgNP labels, a 27-fold increase in signal is observed when a heterobifunctional cross-linker is used to facilitate this labeling. Second, the assay is selective in that it does not cross-react with other cardiac natriuretic peptides. Third, the assay forms in undiluted human serum (though the electrochemical analysis is carried out in buffer). Finally, and most important, the assay is able to detect NT-proBNP at concentrations between 0.58 and 2.33 nM. This performance approaches the critical NT-proBNP concentration threshold often used by physicians for risk stratification purposes: ∼0.116 nM.

Orientation-Controlled Bioconjugation of Antibodies to Silver Nanoparticles.

Here we report on the use of heterobifunctional cross-linkers (HBCLs) to control the number, orientation, and activity of immunoglobulin G antibodies (Abs) conjugated to silver nanoparticles (AgNPs). A hydrazone conjugation method resulted in exclusive modification of the polysaccharide chains present on the fragment crystallizable region of the Abs, leaving the antigen-binding regions accessible. Two HBCLs, each having a hydrazide terminal group, were synthesized and tested for effectiveness. The two HBCLs differed in two respects, however: (1) either a thiol or a dithiolane group was used for attachment to the AgNP; and (2) the spacer arm was either a PEG chain or an alkyl chain. Both cross-linkers immobilized 5 ± 1 Abs on the surface of each 20-nm-diameter AgNP. Electrochemical results, obtained using a half-metalloimmunoassay, proved that Abs conjugated to AgNPs via either of the two HBCLs were 4 times more active than those conjugated by the more common physisorption technique. This finding confirmed that the HBCLs exerted orientational control over the Abs. We also demonstrated that the AgNP-HBCL-Ab conjugates were stable and active for at least 2 weeks. Finally, we found that the stability of the HBCLs themselves was related to the nature of their spacer arms. Specifically, the results showed that the HBCL having the alkyl chain is chemically stable for at least 90 days, making it the preferred cross-linker for bioassays.

Detection of Silver Nanoparticles by Electrochemically Activated Galvanic Exchange.

Here we report on the seemingly simple process of galvanic exchange (GE) between electrogenerated AuCl4- and silver nanoparticles (AgNPs). The results were obtained in the specific context of using AgNPs as labels for bioassays in paper fluidic devices. Results obtained from a combined electrochemistry and microscopy study indicate that the GE process results in recovery of only ∼5% of the total equivalents of Ag present in the system. This low value is a consequence of two factors. First, after an initial fraction of each AgNP undergoes GE, a Au shell forms around the remaining AgNP core preventing further exchange. Second, to simulate a true biological fluid, the experiments were carried out in a Cl--containing buffer. Consequently, some Ag+ formed during GE precipitates as AgCl, and it also serves to block additional GE. Following optimization of the GE process, it was possible to detect AgNP label concentrations as low as 2.6 fM despite these limitations.

Esters of levonorgestrel and etonogestrel intended as single, subcutaneous-injection, long-lasting contraceptives.

An effort with the goal of discovering single-dose, long-lasting (>6 months) injectable contraceptives began using levonorgestrel (LNG)-17-ß esters linked to a sulfonamide function purposed as human carbonic anhydrase II (hCA 2) ligands. One single analog from this first series showed noticeably superior anti-ovulatory activity in murine models, and a subsequent structure-activity relationship (SAR, the relationship between a compound's molecular structure and its biological activity) study based on this compound identified a LNG-phenoxyacetic acid ester analog exhibiting longer anti-ovulatory properties using the murine model at 2 and 4 mg dose than medroxyprogesterone acetate (MPA). The same ester function linked to etonogestrel (ENG) furnished a compound which inhibited ovulation at 2 mg for 60 days, the longest duration of all compounds tested at these doses. By comparison, MPA at the same dose inhibited ovulation for 32 days.