Popcorn‐Shaped Magnetic Core–Plasmonic Shell Multifunctional Nanoparticles for the Targeted Magnetic Separation and Enrichment,Label‐Free SERS Imaging,and Photothermal Destruction of Multidrug‐Resistant Bacteria

Over the last few years, one of the most important and complex problems facing our society is treating infectious diseases caused by multidrug‐resistant bacteria (MDRB), by using current market‐existing antibiotics. Driven by this need, we report for the first time the development of the multifunctional popcorn‐shaped iron magnetic core–gold plasmonic shell nanotechnology‐driven approach for targeted magnetic separation and enrichment, label‐free surface‐enhanced Raman spectroscopy (SERS) detection, and the selective photothermal destruction of MDR Salmonella DT104. Due to the presence of the “lightning‐rod effect”, the core–shell popcorn‐shaped gold‐nanoparticle tips provided a huge field of SERS enhancement. The experimental data show that the M3038 antibody‐conjugated nanoparticles can be used for targeted separation and SERS imaging of MDR Salmonella DT104. A targeted photothermal‐lysis experiment, by using 670 nm light at 1.5 W cm^?2 for 10 min, results in selective and irreparable cellular‐damage to MDR Salmonella. We discuss the possible mechanism and operating principle for the targeted separation, label‐free SERS imaging, and photothermal destruction of MDRB by using the popcorn‐shaped magnetic/plasmonic nanotechnology.     

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small‐Molecule Chemiluminescence Probes

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2–6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy‐dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600‐fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point‐of‐care diagnostics.     

Label‐Free and Ultra‐Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance Spectroscopy

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label‐free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass‐fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 μL volume and label‐free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.     

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.     

Rapid separation and immunoassay for low levels of Salmonella in foods using magnetosome-antibody complex and real-time fluorescence quantitative PCR

A rapid and economical method for detecting Salmonella was developed, based on a novel complex for immunomagnetic separation, which was composed of anti‐Salmonella polyclonal antibody (Ab) and magnetosome (bacterial magnetic particle, BMP) produced by the bacterium Magnetospirillum gryphiswaldense MSR‐1. BMP‐Ab complex was used to capture Salmonella from pure suspensions of S. dublin, S. enteritidis, S. aesch, S. agona, S. abony and S. bareily, from mixed suspensions of S. dublin and Vibrio parahaemolyticus, and from artificially contaminated food samples. Captured Salmonella were then detected by plate count, or real‐time fluorescence quantitative PCR. Capture efficiencies, calculated from plate count, were >80% for the pure Salmonella suspensions of all six strains, and >70% for the mixed suspension. Samples of six food products, with artificial contamination by 6000, 600, 60, or 0.6 cfu/mL S. dublin, were captured by complex and detected by real‐time fluorescence quantitative PCR. Threshold cycle values varied depending on type of food. The lower limit of detectability was 60 cfu/mL without pre‐enrichment, and <0.6 cfu/mL after 3‐h pre‐enrichment. The method described here, based on capture pathogens by BMP‐Ab complex, is sensitive, rapid, and considerably simpler than traditional methods for Salmonella detection. It can be extended to other pathogens by the use of appropriate antibodies.     

Fluorescence Polarization Based Nucleic Acid Testing for Rapid and Cost‐Effective Diagnosis of Infectious Disease

A new nucleic acid detection method was developed for a rapid and cost‐effective diagnosis of infectious disease. This approach relies on the three unique elements: 1) detection probes that regulate DNA polymerase activity in response to the complementary target DNA; 2) universal reporters conjugated with a single fluorophore; and 3) fluorescence polarization (FP) detection. As a proof‐of‐concept, the assay was used to detect and sub‐type Salmonella bacteria with sensitivities down to a single bacterium in less than three hours.     

Effect of size and shape controlled biogenic synthesis of gold nanoparticles and their mode of interactions against food borne bacterial pathogens

In this study, size and shape controlled biogenic synthesis of gold nanoparticles and their antibacterial activity against food borne bacterial pathogens were investigated. Synthesis of gold nanoparticles was carried out using two medicinally important plants Cucurbita pepo and Malva crispa and the size and shape of the nanoparticles were controlled by altering various parameters in the reaction medium. Results obtained from UV–Vis, FE-SEM, EDS and HR-TEM analyses supported the nanoparticles formation. FT-IR analysis confirmed the presence of biomolecules in the plant leaves extracts responsible for reducing and capping agents. Interestingly, the plant extract synthesized gold nanoparticles showed effective inhibition zone against Gram-positive and Gram-negative pathogens. The minimum inhibitory concentration (MIC) of synthesized gold nanoparticles at 400 μg/ml concentration showed effective inhibitory activity against Escherichia coli and Listeria monocytogenes. Conductivity of the medium continuously increased during the nanoparticles treatment with food borne bacterial pathogens resulting in indirect indication of the disruption of bacterial cell membranes. In addition, mode of interactions of gold nanoparticles against food borne bacterial pathogens was demonstrated using Bio-TEM analysis which is clear evident for the disruption of bacterial cell membranes.     

Lateral flow immunodipstick for visual detection of aflatoxin B1 in food using immuno-nanoparticles composed of a silver core and a gold shell

An immunodipstick assay with a lateral flow strip was developed for fast screening of food for aflatoxin B₁ (AFB₁) using the respective monoclonal antibody immobilized on nanoparticles with a silver core and a gold shell (AgAu) as detection reagent. The membrane-based immunodipstick consisted of a test line containing AFB₁ conjugated to bovine serum albumin, and a control line with goat anti-mouse IgG. One to two colored lines are formed on the membrane by using the red AgAu nanoparticles coated with anti-AFB₁ as signaling reagents. Under optimal conditions, the dipstick exhibits a lower visual detection limit of 0.1 ng?mL^?1 of AFB₁. Compared to the use of pure gold nanoparticles, the AgAu nanoparticles strongly enhance the sensitivity of the assay, and the reproducibility and stability are comparable. The assay was evaluated with naturally contaminated samples including rice, wheat, sunflower, cotton, chillies, and almonds, and a good correlation was found with data obtained with a commercially available enzyme-linked immunosorbent assay. The simple and non-instrumental dipstick method may further be extended to the screening of other mycotoxins in food.     

A sandwich electrochemical immunosensor for Salmonella pullorum and Salmonella gallinarum based on a screen-printed carbon electrode modified with an ionic liquid and electrodeposited gold nanoparticles

This article describes an electrochemical immunosensor for rapid determination of Salmonella pullorum and Salmonella gallinarum. The first step in the preparation of the immunosensor involves the electrodeposition of gold nanoparticles used for capturing antibody and enhancing signals. In order to generate a benign microenvironment for the antibody, the ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate was used to modify the surface of a screen-printed carbon electrode (SPCE). The single steps of modification were monitored via cyclic voltammetry and electrochemical impedance spectroscopy. Based on these findings, a sandwich immunoassay was worked out for the two Salmonella species by immobilizing the respective unlabeled antibodies on the SPCE. Following exposure to the analytes, secondary antibody (labeled with HRP) is added to form the sandwich. After adding hydrogen peroxide and thionine, the latter is oxidized and its signal measured via CV. A linear response to the Salmonella species is obtained in the 10⁴ to 10⁹ cfu · mL⁻¹ concentration range, and the detection limits are 3.0 × 10³ cfu · mL⁻¹ for both species (at an SNR of 3). This assay is sensitive, highly specific, acceptably accurate and reproducible. Given its low detection limit, it represents a promising tool for the detection of S. pullorum, S. gallinarum, and - conceivably - of other food-borne pathogens by exchanging the antibody.

We describe an electrochemical sandwich assay based on a screen-printed carbon electrode, gold nanoparticles and ILs and capable of detecting Salmonella pullorum and Salmonella gallinarum. The preparation is outlined in the Schematic.

     

Activation of Nanoparticles by Biosorption for <Emphasis Type="Italic">E. coli</Emphasis> Detection in Milk and Apple Juice

Two types of silver nanoparticles were activated by specific sorption of biomolecules for the detection of Escherichia coli. The capture of this bacterium was performed using polyclonal antibodies (anti-E. coli) biosorbed onto nanospheres or nanorice through a protein-A layer. The bacterial detection was achieved using surface enhancement Raman scattering in order to compare the performance of these two nanoparticles. The activated silver nanospheres showed a better performance mainly due to the dimension of these nanoparticles. The detection limit has been established using the automated Raman mapping system. The technique was capable of detecting 10³ cells/mL in milk and apple juice without any pre-enrichment. With an overall assay time less than 1 h, the process could be easily adapted to detect other pathogens by selecting the pertinent antibody. Furthermore, PCR was used for the DNA verification to assess whether the selected bacterial strain was identical before and after detection.     

Microfluidic based impedance biosensor for pathogens detection in food products

A MEMS‐based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.     

Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 10⁴ cfu.mL^?1 and 10³ cfu.mL^?1, respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods.

Imaging mass spectrometry of gold nanoparticles in a tissue section as an immunohistochemical staining mass probe

For analysis of low abundance peptides in a tissue section, immunohistochemical staining through antibody‐antigen interaction is a usual technique. The antibody is conjugated with a probe moiety that aids in highly sensitive detection. Gold nanoparticles, which show excellent chemical stability and variation of surface modifications, are expected to act as a sensitive mass probe to desorb gold ions (Au⁺, Au₂⁺, Au₃⁺) that are distinguishable from fragment ions from organic molecules. Here, green fluorescent proteins (GFP) in a tissue section of a transgenic zebrafish were detected by the gold mass probe conjugated with antibodies. Due to the efficient ionization and desorption of gold ions, imaging mass spectrometry of Au₂⁺ ions indicated the distribution of gold nanoparticles stained in a tissue section, and the mass signal distribution was consistent with the area where the GFP‐expressing cells were distributed. Conventional immunofluorescence techniques showed intense autofluorescence that come from intrinsic fluorophores in the tissue section. In contrast, the gold nanoparticles acted as an immunostaining mass probe that displayed significantly lower background signals.     

A sensitive liquid chromatography/mass spectrometry‐based assay for quantitation of amino‐containing moieties in lipid A

A novel sensitive liquid chromatography/mass spectrometry‐based assay was developed for the quantitation of aminosugars, including 2‐amino‐2‐deoxyglucose (glucosamine, GlcN), 2‐amino‐2‐deoxygalactose (galactosamine, GalN), and 4‐amino‐4‐deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino‐containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed‐phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10‐fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium‐limited versus magnesium‐replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium‐replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino‐containing moieties in lipid A from a variety of bacterial sources. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a filtration-based SERS mapping platform for specific screening of Salmonella enterica serovar Enteritidis

The presence of Salmonella in natural freshwater and drinking water is a leading cause of intestinal illness all over the world; thus, the detection of Salmonella in water is of great importance to public health. The objective of this study is to develop a rapid screening method for the detection of Salmonella enterica serovar Enteritidis in water involving surface-enhanced Raman spectroscopy (SERS), aptamers, and filtration. SERS offers a great alternative to traditional methods of pathogen detection, with a simplified detection assay and shortened detection time. The specific capturing and labeling of Salmonella Enteritidis are realized by a specific single-stranded DNA aptamer, which is modified with an additional chain of adenine and fluorescein (FAM) and used as presence/absence indicator of Salmonella Enteritidis. By incorporating a vacuum filtration system, bacterial cells recognized by the specific aptamer are concentrated onto a membrane. With additional filtration of gold nanoparticles, the aptamer signals were captured and used to construct a SERS mapping indicating the presence and absence of target bacterial strains with potential quantitative capability. The specificity of the method was validated by using other strains of bacteria such as Escherichia coli and Listeria monocytogenes. The sensitivity of the method goes down to 10³ CFU/mL for 1 mL of sample with a total detection and analyzing time within 3 h. This study demonstrates the capability of the filtration-based SERS platform for detecting Salmonella Enteritidis in various aqueous matrices such as distilled water and rinsing water from fresh produce with high selectivity and sensitivity.

Graphical abstract

     

Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes

A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO₂) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO₂ were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO₂, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 10³ CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 10³ CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.     

Smartphone based bacterial detection using biofunctionalized fluorescent nanoparticles

We are describing immunochromatographic test strips with smart phone-based fluorescence readout. They are intended for use in the detection of the foodborne bacterial pathogens Salmonella spp. and Escherichia coli O157. Silica nanoparticles (SiNPs) were doped with FITC and Ru(bpy), conjugated to the respective antibodies, and then used in a conventional lateral flow immunoassay (LFIA). Fluorescence was recorded by inserting the nitrocellulose strip into a smart phone-based fluorimeter consisting of a light weight (40 g) optical module containing an LED light source, a fluorescence filter set and a lens attached to the integrated camera of the cell phone in order to acquire high-resolution fluorescence images. The images were analysed by exploiting the quick image processing application of the cell phone and enable the detection of pathogens within few minutes. This LFIA is capable of detecting pathogens in concentrations as low as 10⁵ cfu mL⁻¹ directly from test samples without pre-enrichment. The detection is one order of magnitude better compared to gold nanoparticle-based LFIAs under similar condition. The successful combination of fluorescent nanoparticle-based pathogen detection by LFIAs with a smart phone-based detection platform has resulted in a portable device with improved diagnosis features and having potential application in diagnostics and environmental monitoring.

The successful combination of fluorescent nanoparticle-based pathogen detection by lateral flow immunoassay with a smart phone-based detection platform has resulted in a portable device that enables rapid and reliable bacterial detection holding large potential in diagnostics and environmental monitoring

     

Interaction of bacteria and ion‐exchange particles and its potential in separation for matrix‐assisted laser desorption/ionization mass spectrometric identification of bacteria in water

Identification of microbial contaminants in drinking water is a challenge to matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) due to low levels of microorganisms in fresh water. To avoid the time‐consuming culture step of obtaining enough microbial cells for subsequent MALDI‐MS analysis, a combination of membrane filtration and nanoparticles‐ or microparticles‐based magnetic separation is a fast and efficient approach. In this work, the interaction of bacteria and fluidMAG‐PAA, a cation‐exchange superparamagnetic nanomaterial, was investigated by MALDI‐MS analysis and transmission electron microscopy. FluidMAG‐PAA selectively captured cells of Salmonella, Bacillus, Enterococcus and Staphylococcus aureus. This capture was attributed to the aggregation of negatively charged nanoparticles on bacterial cell regional surfaces that bear positive charges. Three types of non‐porous silica‐encapsulated anion‐exchange magnetic microparticles (SiMAG‐Q, SiMAG‐PEI, SiMAG‐DEAE) were capable of concentrating a variety of bacteria, and were compared with silica‐free, smaller fluidMAG particles. Salmonella, Escherichia coli, Enterococcus and other bacteria spiked in aqueous solutions, tap water and reservoir water were separated and concentrated by membrane filtration and magnetic separation based on these ion‐exchange magnetic materials, and then characterized by whole cell MALDI‐MS. By comparing with the mass spectra of the isolates and pure cells, bacteria in fresh water can be rapidly detected at 1 × 10³ colony‐forming units (cfu)/mL. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and characterization of water‐soluble gold nanoparticles stabilized by comb‐shaped copolymers

The water‐soluble gold nanoparticles stabilized by well‐defined comb‐shaped copolymers have been synthesized successfully. The hybrid nanoparticles consist of gold core and poly[poly(ethylene oxide) methyl ether acrylate]‐block‐poly(N‐isopropylacrylamide) [P(A‐MPEO)‐block‐PNIPAM] shell. The water‐soluble comb‐shaped copolymers, P(A‐MPEO)‐block‐PNIPAM with PNIPAM as a handle, were successfully synthesized via a macromonomer technique using reversible addition fragmentation chain transfer (RAFT) polymerization method. The terminal dithioester group of the comb‐shaped copolymer was reduced to a thiol end group forming SH‐terminated copolymers, P(A‐MPEO)‐block‐PNIPAM‐SH. Successively they were used to stabilize gold nanoparticles by the “grafting‐to” approach. The hybrid nanoparticles were characterized by TEM, UV–vis, and HRTEM. Because of the thermosensitive property of PNIPAM in aqueous solution, the comblike copolymer‐tethered gold nanoparticles show a sharp and reversible phase transition at 30 °C in aqueous solution, which was determined by microdifferential scanning calorimetry. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 341–352, 2008     

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection

Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.