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.

Photography was employed for the quantitation and differentiation of G- and V-series nerve agent mimics with the use of self-propagating cascades. Fluoride anion and thiols, released from a G-nerve agent mimic (i.e., diisopropyl fluorophosphate) and a V-nerve agent mimic (i.e., demeton-S-methyl), respectively, were used to initiate self-propagating cascades that amplify fluorescence signals exponentially in a ratiometric manner. A homemade LEGO dark-box, a cell phone, and 96-well plates were employed to collect photographs of the fluorescence response to the analytes. The photographic images were digitally processed in the 1931 xyY color space using a watershed and morphological erosion algorithm to generate chromaticity vs concentration calibration curves. We show that the two different amplification routines are selective for their analyte class and thus successfully discriminated the G- and V-series nerve agent mimics. Further, accurate concentrations of the analytes are determined using the chromaticity and LEGO approach given herein, thus demonstrating a simple and on-site constructible/portable device for use in the field.

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 region 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 halfmetalloimmunoassay, 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 two 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.

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.