Label-free colorimetric estimation of proteins using nanoparticles of silver

Metallic nanoparticles have received considerable attention in bioassays and diagnostics due to their unique surface plasmon resonance (SPR) properties. Gold nanoparticles have been employed for the development of SPR-based colorimetric bioassays. In the present report we have described a sensitive colorimetric approach for estimation of proteins, within a detection limit of 1080 μg/mL, using unmodified silver nanoparticles. Besides the common advantages of colorimetric assay such as simplicity, high sensitivity, and low cost, our method has a label-free design and provides an important and attractive alternative to classical sensing probes and systems. The present work will contribute to the development of nanotechnology-based diagnostic tools.

Owing to inherent photostability, ease of synthesis, biocompatibility, ability to conjugate to biological molecules and innate anti-bacterial as well as anti-platelet properties, nanosilver has established its biomedical potential [11][12][13]. However, though silver nanoparticles possess unique optical properties similar to nanogold, little attention has been paid on nanosilver-based colorimetric assays. Only a few reports are available in literature describing the use of functionalized nanosilver coupled with appropriate ligands in colorimetric detection of DNA, metal ions and proteins [11,[14][15][16][17].
Functionalization of nanosilver can cause its chemical degradation rendering it to be easily oxidized [8]. On the contrary silver nanoparticles have the advantage of higher extinction coefficient as compared to gold particles of comparable sizes.
In our earlier reports we have described synthesis of highly stable, biocompatible nanoparticles of silver and have analyzed their anti-bacterial, anti-platelet and protein stabilizing properties in absence of any modification. Unmodified silver nanoparticles have been shown to be ideal sensors for enzymatic reactions involving dephosphorylation of adenosine triphosphate (ATP) by calf intestine alkaline phosphatase and peptide phosphorylation by protein kinase A [8]. In the present study we describe a sensitive colorimetric assay for quantification of proteins employing unmodified nanoparticles of silver. Our method is based on unique surface plasmon resonance (SPR) of these particles in dispersed and aggregated states and provides a Nano-Micro Lett. 2,

Chemicals and Reagents
Silver nitrate, Sodium hydroxide, sodium chloride, hydrazine, liquid ammonia (30%), and D-glucose were procured from Merck India. Bovine serum albumin (BSA) (fraction V) and immunoglobulin G (IgG) were purchased from Sigma Aldrich. Filters (pore size 0.2 µm) were purchased from Sartorius. All other chemicals were of analytical grade. Milli-Q grade deionized water (Millipore) was used for preparation of the solutions.

Synthesis of silver nanoparticles
Preparation and characterization of highly stable biocompatible nanoparticles of silver have been described in our earlier reports [11][12][13]. Briefly, silver nitrate (17 mg

Absorption spectrophotometry
Absorption spectra of silver nanoparticles, BSA, IgG and conjugates were recorded at wavelengths ranging from 220 to 500 nm in a Beckman spectrophotometer (model DU-640B) equipped with constant temperature cell holder.

Results and Discussion
Nanosilver has propensity to interact with proteins [8,11].
To test that unmodified nanoparticles of silver could act as sensing probes for protein estimation, agglutination of nanosilver was investigated in presence of increasing concentrations of protein (see Fig. 1). BSA was elected as the representative protein for the study due to its well characterized structure and properties as well as its immense physiological significance [18][19][20]. Colloidal solution of 50 μg nanosilver was incubated with increasing concentrations of BSA (0250 μg / ml) and nanoparticle agglutination was induced by NaCl (10 shown to exhibit resistance to salt-induced aggregation in presence of ATP [21]. However, there has been no report describing protein estimation employing unmodified nanoparticles. In order to estimate protein concentration a standard curve of absorbance (at 407 nm) of BSA-nanosilver complex in presence of NaCl versus BSA concentration was generated.
Absorbance was found to increase linearly with BSA concentration in the range from 1080 µg/ml, beyond which it turned into a plateau (see Fig. 2). We were able to determine accurate concentrations of unknown solutions of BSA using this curve. We also obtained a similar curve using another globular protein, IgG, where absorbance (at 407 nm), too, increased linearly over protein concentration 1080 µg/ml (see Fig.3). Subsequently, electron microscopy was performed to examine details of interaction between silver nanoparticles and BSA (see Fig. 4). Spherical, uniformly sized nanoparticles of silver remained well dispersed in solution in the absence of NaCl (see Fig. 4a). Upon exposure to salt aggregates of nanoparticles were evident and free nanoparticles were not visible (see Fig. 4b).
Addition of 1 μg/ml BSA did not bring about any change in agglutination of nanoparticles (see Fig. 4c). However, nanosilver solution supplemented further with increments of BSA (10,50 and 110 µg/ml) exhibited progressive resistance to nanoparticle aggregation (see Fig. 4d-f). Thus, BSA at 10 μg/ml completely prevented aggregation of nanoparticles in response to NaCl treatment and retained uniform distribution of particles identical to nanosilver distribution in absence of salt having enhanced absorbance at 407 nm (see Fig. 4f). Electron microscopic analysis suggests that interaction between proteins and nanosilver prohibits agglutination of latter in response to salt treatment. As inhibition of agglutination is dependent on amount of protein, the characteristic SPR at 407 nm can be used as indicator of protein concentration in the solution.

Conclusions
In the presented report, we have described a sensitive colorimetric approach for estimation of proteins, within a detection limit of 1080 µg/ml, using unmodified nanoparticles of silver. Besides the common advantages of colorimetric assay such as simplicity, high sensitivity, and low cost, our method has