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Peptide-based biosensor utilizing fluorescent gold nanoclusters for detection of Listeria monocytogenes Hanieh Hossein-Nejad-Ariani, Tushar Kim, and Kamaljit Kaur ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00600 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Applied Nano Materials

Peptide-based Biosensor Utilizing Fluorescent Gold Nanoclusters for Detection of Listeria monocytogenes

Hanieh Hossein-Nejad-Ariani, Tushar Kim, Kamaljit Kaur*

Chapman University School of Pharmacy (CUSP), Harry and Diane Rinker Health Science Campus, Chapman University, Irvine, California, 92618-1908, USA

Tel. 714-516-5494; Fax. 714-516-5481 email. [email protected]

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ABSTRACT Listeria monocytogenes is a gram-positive foodborne pathogen that is frequently the cause of listeriosis and meningitis. The high mortality rate (20-40%) from such infections designate this microbe as a high threat to humans. Rapid and specific detection methods for L. monocytogenes can help diminish some of the drawbacks associated with this pathogen. Here we report a novel peptide-based biosensor platform for selective and quick detection of L. monocytogenes species from contaminated food samples. Leucocin A, a potent antimicrobial peptide that displays specific activity against listeria, is utilized to target and bind the receptors present on L. monocytogenes surface. Leucocin A is immobilized on glass surface to obtain a self-assembled monolayer (SAM) of peptide. Peptide SAM is exposed to contaminated sample allowing target bacteria to bind to the immobilized peptide on the surface. The peptide-bound bacteria are then labeled with highly fluorescent gold nanoclusters directly on the glass surface allowing quick detection of bacteria with a limit of detection (LOD) of 2000 cfu in each 10 microliter sample.

The gold nanoclusters are made in situ by directly spotting aqueous

tetrachloroauric acid and 3-mercaptopropionic acid (MPA) on top of bacteria on the glass slide. The MPA-gold nanoclusters thus formed label the bacteria, and absorb in the UV range and emit fluorescence in the visible to near infrared region (λex 304 nm, λem 612 nm). The biosensor assay is portable, simple, fast (45-50 minutes) and can be performed by non-experts, and has the potential to be used as a screening tool for L. monocytogenes in food and pharmaceutical products.

Keywords: Listeria monocytogenes (LM); Biosensor; Leucocin A (LeuA); Gold nanoclusters (AuNCs); Glass surface; peptide self-assembled monolayer

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1.

INTRODUCTION Outbreaks of pathogenic bacteria like Listeria, Escherichia coli and Salmonella in North

America, make clear the danger of microbial pathogens disseminated through contaminated food.1-4 Among these pathogens, L. monocytogenes is a Gram-positive non-spore forming rod bacterium that causes listeriosis in humans, and serious infections manifested by septicemia and meningitis can result in death.1, 2 The ability of this bacterium to grow at temperatures of -0.4 to 45 °C, pH values of 5.0 - 9.6, and in salt concentrations of greater than 10% in the water phase makes it survive in most conditions and accounts for its abundance in nature. Near real-time detection of pathogens like L. monocytogenes would facilitate in minimizing the effects of natural outbreaks or deliberate attacks using infectious agents. Traditional standard microbiological and biochemical assays like agar plates and microbroth dilution assays used to detect and identify pathogenic bacteria are time-consuming and labor-intensive.5 These methods require isolation and/or culturing of large quantities of the infectious agents.

More recently, traditional methods have been replaced with molecular

approaches such as PCR amplification and ELISA based techniques.6

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PCR method enables

high-throughput analysis but has some shortcomings including time consuming and falsepositive results.8

The immunological method utilizes antibodies for selective detection of

pathogens. This technique is specific, sensitive and precise but the use of antibodies makes it comparatively expensive. New detection methods that encompass the accuracy and breadth of traditional microbiological approaches, and improve on the accuracy and sensitivity of molecular approaches would be valuable. Recently biosensor-based methods have gained attention,9-11 where a biosensor utilizes a molecular recognition motif, such as antibody12, 13, carbohydrate 14, 15, aptamer 16, peptide17-22 or

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mixture of these ligands 23, that bind bacteria specifically. The binding is read, to detect bacteria, using different transduction methods that can be categorized into optical, mechanical, electrochemical, potentiometric or impedimetric biosensors. Ahmed et al. reviewed comprehensively biosensors for bacterial detection that do not require sample processing.24 The general characteristics of any biosensor are being time-efficient, compact, portable, sensitive and not labor-intensive. The biosensor approach has been explored for specific detection of L. monocytogenes. 12, 18, 19, 25

Most of the proposed biosensors use antibodies against L. monocytogenes, and include

approaches such as an immunochromatography strip test that specifically detects L. monocytogenes

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, or a multiplex fiber optic biosensor for simultaneous detection of three food

borne pathogens,namely, L. monocytogenes, E. coli O157:H7 and S. enterica. 25 Our group used a listeria-specific antimicrobial peptide, Leucocin A, for specific binding and detection of L. monocytogenes using impedance spectroscopy, microcopy techniques.27,

28

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microcantilever

19, 26

and fluorescence

Antimicrobial peptides (AMPs) are highly specific for pathogenic

bacteria, are easy to synthesize, and have exceptional stability rendering them particularly interesting candidates for use in biological sensor platforms. Several AMPs and peptides without antimicrobial activity have been used as biorecognition probes for the development of peptidebased biosensors.20-22 Leucocin A (LeuA) is a class IIa bacteriocin (also known as pediocin-like bacteriocin) with activity against L. monocytogenes in the nanomolar range.29 Class IIa bacteriocins are small (37-48 amino acid residues) antimicrobial peptides produced by lactic acid bacteria with minimal post translational modifications like disulfide bond formation.30 These cationic peptides have conserved N-terminal sequence (YGNGV) and a variable amphipathic helical C-terminal region.

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The C-terminal region imparts specificity to L. monocytogenes strains and other closely related bacteria. Specifically LeuA binds to the mannose phosphotransferase system (PTS) permease present on the surface of these bacteria.31-33 The activity of bacteriocins vary against different strains of bacteria due to the differential expression levels of the mannose PTS in these strains.33 Class IIa bacteriocins, like LeuA, are exploited for the development of biosensor platforms due to the presence of specific molecular recognition motifs in their sequences.18, 19, 26, 27 Here we have developed a new method for rapid and specific detection of L. monocytogenes by combining the molecular recognition capability of LeuA and fluorescence labelling ability of gold nanoclusters (AuNCs). As mentioned above, LeuA has amphipathic helical motif in the C-terminal region (14-37 residues, Figure 1) which allows specific binding to L. monocytogenes strains. LeuA was immobilized on glass surface to trap target bacteria from a given contaminated sample and the peptide bound bacteria were then labeled with AuNCs for fluorescent detection. Metal nanoclusters and specifically AuNCs have attracted attention as fluorescent agents due to several characteristics including excellent photo-stability, biocompatibility and ultra-small size (95% pure, and were obtained in good yields (LeuA 60% and negative peptide 75%). LeuA and negative peptide were checked for activity using agar plate inoculated with L. monocytogenes Type I. Briefly, TSB agar plate was spotted with different concentrations of peptide (2.3, 0.23, and 0.023 mM, dissolved in water) with a spot volume of 10 µL. Then the 6 mL soft agar inoculated with 60 µL L. monocytogenes (overnight culture, undiluted) was plated over the TSB agar.43 The plate was incubated at 37 °C and zone of inhibition was checked after 24 hours.

2.5.

Peptide immobilization. Two different methods were evaluated for immobilization of

peptide on glass surface. A FITC labeled 11-mer peptide (FITC-WxEAAYQkFLA) was used as a representative peptide to determine the immobilization method. In the first approach, peptide

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was covalently immobilized on the glass surface (glass slide, Fisher Scientific, L x W: 75 x 25 mm).44 For this method, first the glass surface was etched using piranha solution (3 parts sulfuric acid with 1 part 30% hydrogen peroxide) for 20 min. After washing with copious amount of MillQ water, the glass surface was modified with (3-aminopropyl)triethyloxysilane (APTES, 2%, v/v) solution. The slide was dipped in 2% APTES in pre-heated toluene solution for 1 hour. After which the slide was dried on a hot surface (80 °C) for 1 hour. Finally, the slide was treated with 2.5% glutaraldehyde in PBS for 20 minutes. The glutaraldehyde acts as a linker for the peptide immobilization. FITC (10 µL) labeled peptide was then spotted on the functionalized slide. After air drying, the slide was washed for 20 seconds using MillQ water. The second approach was based on non-covalent immobilization of peptide on the glass slide.45, 46 FITC labelled peptide was spotted directly on the glass slide surface (10 µL) and was left to dry. Then the slide was washed with MillQ water for 20 seconds. In both experiments (covalent and non-covalent) six identical spots were made, and the fluorescence was quantified using Chemidoc imager. The average fluorescence intensity was calculated for each method.

2.6.

Detecting L. monocytogenes using AuNCs on glass surface. Aqueous solution of LeuA

(10 µL, 0.1 mM) was spotted on glass slide (~4 mm diameter) in triplicates. The peptide spots were allowed to air dry (~ 20 min) followed by spotting bacteria L. monocytogenes ATCC 43256 (10 µL, 107 cfu) or no bacteria (10 µL water) on top of the peptide spot. The glass slide was then dipped in MillQ water for 10 seconds to remove all non-specific interaction of bacteria with the peptide. The slide was allowed to air dry and finally MPA-AuNCs (9 µL, 10 mM HAuCl4 and 3 µL, 100 mM MPA) were spotted. MPA-AuNCs (12 µL) alone were also spotted as a control.

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The mean fluorescence intensity was recorded using Chemidoc imager. The experiment was repeated three times.

2.7.

Optimization of peptide concentration. The bio-sensor was optimized by evaluating

different LeuA concentrations on glass surface. The slide was spotted with 10 µL of varying peptide concentrations (0.05-0.4 mM). The peptide spot was allowed to air dry for 20 min. Next bacteria (L. monocytogenes ATCC 43256) was spotted at a constant concentration (10 µL, 104 cfu). After drying, the slides were dipped in MillQ water for 10 seconds. Lastly MPA-AuNCs (12 µl) were spotted to quantify the fluorescence intensity of the spots.

2.8.

Sensitivity and selectivity of the biosensor. To obtain the limit of detection (LOD), the

biosensor assay was performed with varying number of bacteria in 10 µL sample. An overnight culture of L. monocytogenes ATCC 19116 was diluted to obtain samples with 0, 200, 1000, 2000, and 10000 cfu/10 µL. Glass slide was prepared with peptide spots (LeuA, 0.1 mM, 10 µL) as descried above and bacteria (10 µL) were spotted on the peptide spot followed by MPAAuNC labeling and fluorescence recording using Chemidoc imager. The selectivity of the biosensor assay was tested with three L. monocytogenes strains (ATCC 19116, ATCC 43256 and Pat Type I), and two other Gram-positive strains (Bacillus cereus ATCC 14579 and Staphylococcus aureus ATCC 29213). Gram-negative Salmonella enterica and Escherichia coli DHα were also used. Water (no bacteria) and 24-mer negative peptide with L. monocytogenes 19116 were used as controls. performed as described above.

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The biosensor assay was

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2.9.

Detection of L. monocytogenes in spiked milk sample. A spiked milk sample was

prepared using milk (3.5% fat, 1 mL) diluted with water (9 mL). L. monocytogenes 19116 was added to the diluted milk to obtain a bacterial concentration of 104 cfu/10 µL. Water was used as a control. The biosensor assay was performed on spiked milk sample as described above.

2.10.

Statistical analysis. Data are presented as mean ± standard deviation (SD) of triplicate

measurements throughout the manuscript. Statistical significance of difference was evaluated using the Student’s t-test or one way ANOVA test. The significance level was set at 0.05. All data were processed using ORIGIN 2016 (academic) software.

3.

RESULTS AND DISCUSSION

3.1.

Preparation of AuNCs using MPA as a ligand. Several synthetic methods have been

introduced recently for the synthesis of AuNCs.37, 40, 42, 47, 48 The most popular method is where AuIII is reduced to AuI using different ligands, at times accompanied by a reducing agent such as NaBH4. Ligands that have been used to stabilize nanoclusters include biomolecules or small thiol-containing molecules like MPA and 6-mercapto-hexanoic acid (MHA). These ligands also serve as reducing agents to help convert gold ions into gold nanoclusters. Here we synthesized AuNCs using HAuCl4 with 3-MPA as the stabilizer or ligand.42 Thiol containing molecules like 3-MPA are the most commonly used stabilizers for both nanoclusters and nanoparticles as this strategy allows one-pot facile synthesis.47

Simply a

solution of HAuCl4 was mixed with 3-MPA in about 1:3 molar ratio and the synthesized MPAAuNCs were characterized using fluorescence spectroscopy.

As shown in Figure 2A, the

AuNCs showed maximum absorption at ~304 nm with emission around 612 nm.

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The


nanoclusters displayed light yellow color under visible light, however when excited under UV light AuNCs emitted red

(A)

(B)

Figure 2. Characterization of MPA-AuNCs. (A) UV-visible absorption spectra of AuNCs. (B) Fluorescence spectra (λex/λem 304/612 nm) of MPA-AuNCs with respect to reaction time (0, 0.5, 2, 4, 6, and 21 h).

fluorescence. The emission from the AuNCs was monitored overtime to evaluate their stability at room temperature. As shown in Figure 2B, the AuNCs were stable up to 21 hours with fluorescence being highest at ~4 hours. The TEM and HRTEM images of similar MPA-AuNCs have been reported previously.42

Further characterization was done using MALDI-TOF mass

spectrometry as mass spectrometric analysis is important for the precise determination of the cluster size. It allows determination of not only the metal atoms but also the protecting ligands present in the AuNCs. The mass spectrum repeatedly showed a dominant peak for AuNCs at

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1230.5 Da, followed with 1246.4 Da (SI Figure S2). These peaks at 1230.5 Da and 1246.4 Da were calculated to be AuNCs composed of 4 Au and 4 MPA atoms with sodium [Au4(MPA)4+Na] and potassium [Au4(MPA)4+K], respectively. Most detection techniques that use gold nanoclusters either involve long and complicated methods for preparation of AuNCs or sometimes give clusters that are not stable over time.14, 49-51 The MPA-AuNCs prepared here were easy to synthesize and were stable with excellent emission properties. These AuNCs were stable for a week at room temperature and for few weeks in the refrigerator (data not shown).

3.2.

Peptide immobilization on glass surface.

We previously showed that class IIa

bacteriocin LeuA (37-mer) or fragment (24-mer, 14-37 residues, Figure 1) derived from the Cterminal amphipathic helical region of LeuA bind specifically to L. monocytogenes and closely related Gram-positive strains

18, 26, 27

. In these studies, the peptide was covalently immobilized

on surface, and the binding between the peptide and bacteria was sensed using fluorescence microscopy (using bacteria labeled with fluorescent dyes like CyQuant and propidium iodide)28, microcantilever bending (label-free detection)

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or impedance spectroscopy (label-free

detection).18 In this study, we have used full length LeuA for binding L. monocytogenes. A short sequence (20-mer, 5-24 residues, Figure 1) derived from the middle region of LeuA which lacks binding to L. monocytogenes 26 was designed and used as a negative control peptide. Previously we found by screening of a peptide library of 14-mer overlapping sequences from LeuA that residues 5-24 of LeuA had no binding to the L. monocytogenes strains.26 Also we substituted the two cysteine residues (9 and 14) in the N-terminal region, which are important for LeuA activity, with alanine in the control peptide. Using bioactivity plate assay it was confirmed that LeuA

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was active against L. monocytogenes, whereas, the control 20-residue sequence was inactive. To obtain a self-assembled monolayer (SAM) of peptide on glass surface for the biosensor assay, both non-covalent and covalent immobilization methods were evaluated. For the non-covalent method, the peptide was immobilized by spotting a small aliquot (10 µL) of peptide solution directly on the glass surface. Whereas, the covalent immobilization involved several steps, namely, etching the glass, modifying the surface with APTES followed by treatment with glutaraldehyde and the peptide (SI Figure S3a). A FITC-labeled peptide was immobilized using both the non-covalent and covalent methods, and the mean fluorescence intensity (MFI) of the surface bound peptide was compared (SI Figure S3b).

We found that non-covalent

immobilization was fast and easy, and deposited about similar amount of peptide as the covalent immobilization. The average MFIs of the spots (six) for the non-covalent and covalent methods were 19708 ± 3299, and 23927 ± 4902, respectively, suggesting similar peptide density (average MFI was not significantly different) upon immobilization. For all subsequent experiments, noncovalent immobilization of peptide on glass surface was used. The peptide immobilized glass slides were stored in refrigerator or freezer (-20 °C) until use. Most studies utilize a covalent bond between activated factors on surface and amino acid group of peptide or protein for immobilization. While the covalent bond make the sensor stable for a longer time

44, 52

, the long and sophisticated steps to immobilize the peptide/protein is one

of the disadvantage of using this method. The non-covalent interaction of protein with glass surfaces has also been studied.45,

46

Kristensen’s group showed rapid adsorption of cationic

membrane-active peptides to plastic ware and glassware surfaces.45 It is suggested that there are at least two adsorption interactions possible between the glass and the protein.45,

46

The first

interaction is fast and mainly involves the silanol-ionic amine bonding and the hydrogen bond.

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The second interaction which is much slower depends on the protein’s molecular weight and is dictated by the capability of protein to diffuse into the porous glass.

3.3.

Labeling bacteria with AuNCs. Surface bound bacteria were labelled with MPA-

AuNCs for easy detection using fluorescence. In order to maximize the labeling (fluorescence) using MPA-AuNCs, the concentration of MPA-AuNCs used to label bacteria was optimized. First different concentrations of HAuCl4 (1 mM, 10 mM, and 100 mM) or MPA (10 mM and 100 mM) were used to obtain high fluorescence of labelled bacteria compared to no bacteria (MPAAuNCs alone). We found that MPA-AuNCs prepared with 10 mM HAuCl4 (10 µL) and 100 mM MPA (3 µL) displayed higher fluorescence compared to other concentrations used (SI Figure S4). The fluorescence was further optimized by evaluating different amount (volume) of HAuCl4 (3, 5, 9 or 11 µL; 10 mM) and MPA (1, 3, 5, or 9 µL: 100 mM) spotted on bacteria. As shown in SI Figure S5, bacteria labeled with 9 µL HAuCl4 (10 mM) and 3 µL MPA (100 mM) showed highest fluorescence difference between the bacteria and no bacteria samples. This concentration of HAuCl4 (9 µL, 10 mM) and MPA (3 µL, 100 mM) was used for all subsequent experiments. Interestingly the molar ratio of HAuCl4 to MPA remained similar (~ 3:1) to the one used for making AuNCs in solution (Figure 2). Confocal microscopy was used to image bacteria labelled with MPA-AuNCs. Non-pathogenic Listeria innocua (which are very similar to pathogenic L. monocytogenes) were used for confocal experiments and were spotted on a glass slide followed by labeling with MPA-AuNCs (12 µL). The bacteria were also co-stained with DAPI before imaging using confocal microscope. The images (Figure 3 A-C) clearly show that all bacteria were uniformly labeled with DAPI and AuNCs.

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Finally bacteria (104 CFU/mL) were labelled with varied concentration of MPA-AuNCs. A stock solution of MPA-AuNCs was diluted with water and spotted on bacteria on the glass slide followed by fluorescence imaging using Chemidoc imager for about 4 hours at regular intervals.

It was observed (Figure 3 D) that the fluorescence increased with increasing

concentration of MPA-AuNCs and saturated when the gold nanocluster concentration was in the range of 9-15 µL. The fluorescence intensity of labeled bacteria was maximum at ~50 mins, thereafter dropped a little bit at ~ 2 hours and remained the same for up to 4 hours. The exact interaction between AuNCs and bacteria is not yet clear. Recent studies highlight that the ligand plays a major role in the specific interaction between AuNCs and bacteria.50,

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For instance, Cheng et al. showed that AuNCs made with vancomycin

(AuNC@Van) bind on the surface of Gram-positive Staphylococcus aureus by interaction between the vancomycin (the ligand) and the peptidoglycan of Gram-positive bacteria. Vancomycin specifically binds the D-ala D-ala of N-acetylglucosamine and N-acetylmuramic acid (NAG-NAM) peptide subunits of the bacterial cell wall peptidoglycan.50 The authors found no such binding of AuNC@Van when the experiment was repeated with gram-negative E. coli which do not have peptidoglycan on the surface. Also Mukherji et al. selectively probed some receptors on bacteria using Au nanoclusters.53 For MPA-AuNCs, our conjecture is that the MPA-AuNCs interact with bacteria via the protein thiol groups on the surface of bacteria and the gold of AuNCs due to the strong gold-thiol interactions. The MPA ligand is mainly involved in nanocluster stabilization.

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Figure 3. (A-C) Confocal fluorescence microscopy images of Listeria innocua ATCC 33091 after labelling with MPA-AuNCs and nuclear stain DAPI. (A) shows emission for DAPI at 396485 nm, (B) shows emission for MPA-AuNCs at 588-695 nm, and (C) is an overlay of A and B. Scale bar = 1 µm (D) Fluorescence of L. monocytogenes 43256 (104 CFU/mL) after labeling with increasing concentration of MPA-AuNCs (0 – 15 µL). The fluorescence was imaged using ChemiDoc imager at four different time intervals (50, 140, 180, and 260 minutes) after labeling the bacteria on the glass slide.

Next, MPA-AuNCs were used to label bacteria that bound to peptide to develop the biosensor assay. A glass slide was spotted with peptide (LeuA), and bacteria or no bacteria (water) were spotted on the peptide. Finally, the spots were labelled with MPA-AuNCs by adding HAuCl4 and MPA. AuNCs were also spotted alone for comparison of fluorescence between the AuNCs alone and AuNCs labelled bacteria. As shown in Figure 4, AuNCs labeled bacteria showed significantly higher fluorescence (MFI 62390 ± 5975) compared to AuNCs alone (MFI 31860 ± 11608). Also, AuNCs addition to peptide spot showed no increase in

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AuNCs fluorescence (MFI 34352 ± 1812). It was observed that the peptide or bacteria show no auto-fluorescence at >600 nm.

Figure 4. Comparison of AuNCs fluorescence on glass surface. Defined spots (with 4 mm diameter) on glass surface are prepared by spotting (i) AuNC (12 µL) alone, (ii) LeuA peptide (10 µL, 0.13 mM) followed by AuNCs (12 µL), and (iii) LeuA (10 µL, 0.13 mM) followed by bacteria LM (10 µL, 107 cfu) and then AuNCs (12 µL). Each spot is allowed to dry before spotting the next. In addition, peptide (LeuA) and bacteria (LM) were also spotted alone as controls. LM stands for Listeria monocytogenes ATCC 43256.

3.4.

Assay optimization. In order to trap higher number of bacteria from the sample to

increase the limit of detection, we optimized the peptide concentration for the peptide SAM at each spot on the glass slide. The slide was spotted (10 µL) with varying peptide concentration (0.05-0.4 mM), and the bound bacteria from the sample (104 cfu/10 µL) was estimated based on the fluorescence intensity of each spot. The results show increased fluorescence intensity with increase in peptide concentration up to 0.1 mM, after which there was a small drop in fluorescence for 0.2-0.4 mM concentrations (Figure 5). The spot with a peptide concentration of

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0.1 mM showed the highest fluorescence (21275 ± 2177) and this peptide concentration was used for all subsequent experiments.

Figure 5. Fluorescence response of bound bacteria with increasing LeuA peptide concentration per spot. LeuA (10 µL) was spotted on glass slide followed by L. monocytogenes ATCC 43256 (104 cfu/10 µL) and AuNC (12 µL). The fluorescence intensity of AuNC labelled bacteria was read using Bio-Rad Chemidoc imager.

3.5.

Estimation of the limit of detection. To obtain the LOD of the peptide-based biosensor

assay, LeuA (0.1 mM) was dry spotted on slides with varying bacterial concentration (0, 200, 1000, 2000, and 10000 cfu/10 µL), after which AuNCs were applied to label the bound bacteria. As shown in Figure 6, sample containing 2000 cfu or higher per spot showed significantly higher fluorescence compared to the water alone. Sample containing 10000 cfu bacteria showed similar fluorescence (30842 ± 4696) as the 2000 cfu sample (31473 ± 2932) suggesting that the peptide spot was saturated with bacteria and did not allow binding of any additional bacteria above 2000 cfu. From these results, it can be estimated that the LOD for the bio-sensor is around 2000 cfu/10 µL or 2 x 105 cfu/mL.

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Previously we detected bacteria at a similar concentration of 105 cfu/mL using the microcantilever method26 and 106 cfu/mL using CyQuant labeled bacteria,27 while other methods allowed detection at much lower concentrations. For instance, with impedance spectroscopy18 and more recently with a biomaterial microcantilever19 we achieved a LOD of 103 cfu/mL. In addition, labelling peptide bound bacteria with propidium iodide and detection with confocal microscopy allowed detection at 103 cfu/mL.28

However, most of these methods require

sophisticated equipment like microcantilever or confocal microscopy which mandates trained personnel.

Figure 6. Fluorescence response of bound bacteria with increasing bacterial concentration (L. monocytogenes ATCC 19116) per spot. Peptide LeuA (10 µL, 0.1 mM) was spotted on glass slide followed by bacteria (10 µL) and MPA-AuNCs (12 µL). The fluorescence intensity of labelled bacteria was read using Bio-Rad Chemidoc imager. Statistical significance was denoted by * (P

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