Elsevier

Biosensors and Bioelectronics

Volume 53, 15 March 2014, Pages 519-527
Biosensors and Bioelectronics

A filter-like AuNPs@MS SERS substrate for Staphylococcus aureus detection

https://doi.org/10.1016/j.bios.2013.10.017Get rights and content

Highlights

  • SERS filter-like substrate made of AuNPs embedded in mesoporous silica (denoted as AuNPs@MS) was applied for SERS applications.

  • Staphylococcus aureus can be concentrated on the filter-like AuNPs@MS substrates within a few seconds.

  • SERS spectra showed more characteristic peaks with significant enhancement on the spectra signals.

  • Highly reproducible SERS spectra can be obtained from the substrate.

  • About 900 times of SERS enhancement can be achieved (Au wt%=16) compared with Raman spectra obtained from the pure mesoporous silica (Au wt%=0).

Abstract

An accurate, highly sensitive and rapid identification assay of cells is extremely important in areas such as medical diagnosis, biological research, and environmental monitoring. Laboratory examinations of clinical isolates require time-consuming and complex processes to identify the colony count, with approximately 106–108 cells needed for the characterization of strains. In the present study, a highly sensitive SERS filter-like substrate is prepared with AuNPs embedded in mesoporous silica (denoted as AuNPs@MS) synthesized by a simple one-spot method, and an example of its use for the filtration and concentration of analytes from aqueous samples is reported. In an application for Staphylococcus aureus SERS discrimination, the results show that the target cells can be concentrated on the filter-like AuNPs@MS substrates within a few seconds, with much better reproducibility with regard to the SERS spectra that are obtained. The experimental findings suggest that the AuNPs@MS substrate supports much higher intensity with more distinguishable peaks compared to Au/Cr-coated substrate, and the reproducibility is also significantly improved. The substrates investigated in this study generated 900 times more SERS signals at a concentration of 106 CFU/mL in the detection of S. aureus on mesoporous silica (Au wt%=0) when using AuNPs@MS with 16 wt% AuNPs. The limitation of this filter-like SERS substrate can be applicable for small volume samples (few to hundred microliter).

Introduction

There is increasing interest in the detection of certain bacteria due to the many essential functions these biological systems have in nature, as well the negative effects that some can have on animals, humans and the environment. The accurate, highly sensitive and rapid identification assay of cells is thus extremely important in areas such as medical diagnosis, biological research, and environmental monitoring. Laboratory examinations of clinical isolates, like Staphylococcus aureus (S. aureus) from bodily fluids and Helicobacter pylori (H. pylori) from gastric disease or duodenal ulcer specimens, currently require highly time-consuming and complex processes to identify the colony count, and approximately 106–108 cells are needed for the characterization of strains (Guruge et al., 1998, Prinz et al., 2001, Yang et al., 1997). Developments in analytical instrumentation over the last two decades have utilized a number of different physico-chemical spectroscopic methods, the most common of which are pyrolysis mass spectrometry (PyMS) (Barshick et al., 1999, Timmins et al., 1998), Fourier-transform infrared spectroscopy (FT-IR) (Edwards et al., 1995, Naumann et al., 1995, Williams and Edwards, 1994) and UV resonance Raman spectroscopy (Ghiamati et al., 1992, Maquelin et al., 2002, Maquelin et al., 2000, Nelson et al., 1992). When applied to complex biological samples, such as cells or tissues, the individual spectra contributions of the molecular species accumulate to reflect the overall molecular composition. Such spectra have been shown to be highly suitable for rapid identification of bacteria (Ibelings et al., 2005, Udelhoven et al., 2000) and yeasts (Freydiere et al., 2001, Tintelnot et al., 2000), because of the reproducible and distinctive characterization of different species. Raman spectroscopy is thus often referred to as ‘whole-organism fingerprinting’, since it is able to give comprehensive, quantitative information about the overall biochemical composition of a microbial sample (Magee, 1993).

Surface Enhanced Raman Scattering (SERS), using photons as probes (i.e. Raman-active analyte molecules), results in higher sensitivity and resolution spectra without damage to samples, and is thus an attractive approach for chemical and biological analyses. In order to obtain greater sensitivity and signal enhancement, SERS-active substrates have been designed and prepared using polymerization, surface modification or immobilization, and ether electrochemical deposition of metal particles (or colloids) to create nanoparticles or a roughs metal surface with various geometric forms under different matrices (Luo and Fang, 2005a). These special particle surfaces can be applied for the discrimination of biological molecules (Grow et al., 2003; Lin et al., 2008), viruses (Shanmukh et al., 2006), yeasts or bacteria (Jarvis and Goodacre, 2004, Kahraman et al., 2008) by direct contact with the surface of these targets. The enhancement factor with this approach can be as much as 1014–1015, which means it is sensitive at the molecular level (Rösch et al., 2003, Schuster et al., 2000, Xie and Li, 2003). Other SERS spectra-based assays have been reported for pathogen analysis in metal particle solutions, i.e. gold and silver (Guicheteau et al., 2008), and by placing bacteria directly on a gold nanoparticle (~80 nm) covered SiO2 SERS chip (Premasiri et al., 2005), on a patterned gold nanohole and nanodisk surface with precisely controlled size and spacing (Wang et al., 2006a, Yu et al., 2008), or on an electrochemically roughened metal surface with immobilized capture biomolecules (Grow et al., 2003). These methods all show that significant signal enhancement (normally over 104), with an increased number of distinguishable peaks, relative peak intensities, appreciable spectra, and better species differentiation, can be obtained in bacteria SERS spectra than with normal Raman (non-enhanced) spectra.

Based on these results, Huang et al. used a heat-induced SERS sensing method to directly dry a glutathione-AgNPs mixture, and found a detection limit of ca. 50 nM with an enhancement factor of about 7.5×106 within 5 min (Huang et al., 2009). However, aggregation of the analyte and colloid particles may inhibit the effectiveness of SERS, as Moskovits showed that only the gaps between the cellular and metal surfaces at the nanoscale size can contribute to SERS spectra (Kneipp et al., 2006, Moskovits, 1985).

Concentrating the analyte for biological analysis by a process of water evaporation is a time consuming processes. Ota et al. dropped silver colloid solution containing l-phenylalanine on to a filter-paper and measured the spectra by near-infrared SERS (NIR-SERS) with the excitation set at 1064 nm. Their results showed the maximum enhancement factor of l-phenylalanine is only about 30 (Ota et al., 1997). Many similar studies can be found in the literature, such as Luo and Fang, who proposed a new SERS substrate based on dried gold nanoparticle coated filter paper or filter film in order to carry out SERS of C60/C70. They reported an enhancement factor of up to ~105, as this approach avoided the influence of solvents in the C60/C70 solution and water in gold hydrosols (Luo and Fang, 2005). Cheng et al. deposited silver nanoparticles directly on commercially available filter paper, and, compared to glass-based SERS substrates, 50 times more SERS signals were observed in the detection of tyrosine, and they calculated the enhancement factor could be over 107, giving a linear range of up to 100 μM with a detection limit of 625 nM. Their substrates thus provide much higher SERS signals and greater reproducibility for detection of tyrosine in aqueous solution (Cheng et al., 2011). Other filter-based SERS substrates coated with silver or gold nanoparticles have been reported for the characterization of chemical molecules (Bhandari et al., 2009, Laserna et al., 1989, Wu and Fang, 2003, Wu and Fang, 2004) or environmental contaminants (Bhandari et al., 2009). For samples with high water content, such as those found in microorganism measurements, filter-like substrates provide rapid water adsorption or faster filtration ability, which can be very advantageous with regard to increasing the concentration of targets (Cheng et al., 2011, Xie et al., 2012).

In general, SERS is a powerful tool for the characterization of the chemical structure or composition of materials, such as biological samples. However, some problems with regard to using SERS substrates need to be overcome, such as the use of a protecting agent or surfactant, which is often required to prevent the aggregation of nanoparticles during the preparation process, and which also generate Raman signals, making it more difficult to recognize those from the analyte. Another problem is that the adsorption of analytes in the mixture is non-uniform, because the structure of prepared nanoparticles is not well-defined, and thus the maximum enhancement of SERS signals cannot be achieved. As previously reported, the distance between nanoparticles and bacteria may be longer than a few nanometers, and this can be outside the SERS hot-spot (Kneipp et al., 1996, Nie and Emory, 1997, Xu et al., 1999). Based on a review of the literature, some of the improvements that are needed with regard to SERS-active nanoparticles are as follows: (1) the ability to increase the adsorption or affinity in relation to the biological sample; (2) the magnitude of the enhancement of the resonance effect with regard to the Raman signals; (3) the development of novel nanoparticles that can be used without a protecting agent; (4) reducing the fluorescence “quenching” effect; and (5) the capability to collect targets from low concentration liquid samples.

Mesoporous silicas with a high surface area (more than 300 m2/g), large pore volume, and adjustable pore size (1.5–50.0 nm) have attracted much attention from the chemical, bio-chemical and material community (Kresge et al., 1992). Silica-based surfaces provide excellent matrices for biomolecule attachment, by direct, non-direct, or covalent methods, as report by Doering et al. (2007) According to silica chemistry, mesoporous silicas have both high stability and low bio-toxicity, and thus can be considered as a superior solid matrices for the incorporation of nanoparticles and catalytic reaction sites (Lin and Mou, 2000, Ying et al., 1999, Zhao et al., 1998). In addition to using cationic surfactants and block copolymers as templates, gelatin is a natural polymer with many amine functional groups which can have a high affinity to strongly interact with silanol groups on the silicate species via multiple hydrogen bonds (Iler, 1979). Moreover, its functional groups (i.e., –NH2, –S–CH3) can also act as the protecting agent of the noble metal nanoparticles. As previously reported by Brayner et al. and our team, a simple method for the preparation of porous silica can be achieved by using gelatin, which acts as the organic template and the protecting agent of the embedded metal nanoparticles (Brayner et al., 2005, Lin et al., 2007).

In this work, we demonstrate the fabrication and application of a filter-like bioactive SERS substrate made of gold nanoparticles@mesoporous silica (AuNPs@MS), as well as the SERS measurement of bacteria signals generated from developed-substrates by adding a micro-liter sample. The SERS active abilities (Au nanoparticles) are analyzed, and the results show that this approach can produce strong SERS signals with good bacterial discrimination without any need for pre-labeling.

Section snippets

Synthesis of gold nanoparticle embedded mesoporous silica

To produce noble AuNPs@MS, we previously proposed a simple one-pot synthesis method for the preparation of AuNPs@MS powder, as illustrated in Fig. 1 (Lin and Mou, 2000, Lin et al., 2007). Briefly, 50.0 mL of 1 mM salt (HAuCl4) aqueous solution was slowly added to a mixture of 0.5 g of gelatin and 15.0 g of H2O and gently stirred to form a clear solution at 4 °C. 5.0 mL of NaBH4 aqueous solution (containing 10 mg NaBH4) was then added dropwise, and this led to the formation of an Au-nanoparticle

Properties of AuNPs@MS

In this work, the synthesis procedures of the AuNPs@MS using as surface enhanced Raman scattering substrate is shown in Fig. 1. Fig. 2 shows the TEM images of the AuNPs@MS samples with different Au nanoparticles contents, and the good dispersion of the Au NPs in the mesoporous silica matrixes can be clearly seen. As previously reported (Cheng et al., 2010, Luo and Fang, 2005), the surface roughness and size of metal particles both act as important factors in the development of SERS substrates.

Conclusions

This work provides a new and convenient one-pot method to directly synthesize a noble AuNPs@MS substrate with a high surface area for SERS applications. Highly reproducible SERS spectra can be obtained from the substrate with significant enhancement on the spectra signals. It was used for the discrimination of the S. aureus SERS fingerprint, and the results showed that the AuNPs@MS substrate produce a much higher intensity and more distinguishable peaks of bacteria spectra when compared with

Acknowledgements

This study is co-financed by the National Science Council of the Republic of China (99-2218-E-029-004, 100-2320-B-029-001). We also thank the Multidisciplinary Center of Excellence for Clinical Trial and Research for its financial support for this project (DOH102-TD-B-111-002), Department of Health, Executive Yuan, Taiwan.

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