Highly sensitive optical biosensing of Staphylococcus aureus with an antibody/metal–organic framework bioconjugate

Neha Bhardwaj ab, Sanjeev K. Bhardwaj ab, Deepanshu Bhatt ab, Satish K. Tuteja c, Ki-Hyun Kim *d and Akash Deep *ab
aCentral Scientific Instruments Organisation (CSIR-CSIO), Sector 30 C, Chandigarh, 160030, India. E-mail: dr.akashdeep@csio.res.in
bAcademy of Scientific and Innovative Research, CSIR-CSIO, Sector 30 C, Chandigarh, India
cIWE1-Institut für Werkstoffe der Elektrotechnik RWTH Aachen University, Germany
dDepartment of Civil and Environmental Engineering, Hanyang University, 222 Wangsimni-Ro, Seoul 04763, Korea. E-mail: kkim61@hanyang.ac.kr

Received 13th November 2018 , Accepted 5th January 2019

First published on 7th January 2019

In this research, a new luminescent bioprobe was developed for the detection of S. aureus based on bio-conjugation of an amine functionalized metal–organic framework (NH2-MIL-53(Fe)) with an anti-S. aureus antibody (Ab). The formation of the desired bioprobe (Ab/NH2-MIL-53), in its liquid phase, has been verified with several spectroscopic and structural characterization techniques. The bioprobe was incubated with varying concentrations of S. aureus bacteria. The resulting antibody conjugated bioprobe (Ab/NH2-MIL-53) maintained a strong inverse correlation in which decreases in the fluorescence intensities were accompanied by an increase in the bacterial count. Thus, the potential of the herein developed probe has been successfully demonstrated for the detection of S. aureus with a low limit of detection (85 CFU mL−1) over a wide concentration range (4 × 102–4 × 108 CFU mL−1). It was further found to be reliable with regard to inter-/intra- precision assays and long-term stability. The feasibility of the method was further supported through the detection of S. aureus spiked in environmental samples (e.g., river water and cream pastry).

1. Introduction

The consumption of bacteria contaminated foods is a serious health concern worldwide.1–3 The majority of bacterial illnesses may reflect the consequences of an infection or intoxication from diverse species of bacteria such as Staphylococcus aureus (S. aureus), Salmonella typhimurium (S. typhimurium), Escherichia coli O157[thin space (1/6-em)]:[thin space (1/6-em)]H7 (E. coli), Listeria monocytogenes (L. monocytogenes), Streptococcal sp., Clostridium perfringens (C. perfringens), Campylobacter jejuni (C. jejuni), Shigella sp., and Bacillus cereus (B. cereus). Among the above food-borne pathogens, S. aureus is known to cause a wide range of diseases such as dermatitis, food poisoning, and gastrointestinal tract infections. S. aureus has the ability to produce heat-stable entero-toxins and to increase the antibiotic resistance (especially methicillin resistant S. aureus, MRSA).3–5 Several cases of MRSA outbreaks have been reported in recent years to stress a serious necessity towards measures for control and prevention of S. aureus.1

Generally, the food industries rely upon chemical and microbiological techniques to assess their product quality. Traditional culture-based methods, including microbiological and biochemical analyses, are still the most practiced options.6 The polymerase chain reaction (PCR) is another popular method used for the quantitative determination of bacteria. Despite their proven utility, the above mentioned conventional methods are time-consuming and labour intensive. To practice these methods, a single bacterial cell must be grown into a visible colony to amplify the signal.3 The applications of such techniques are rather inconvenient in situations demanding more frequent, rapid, and on-site screening of the pathogen contamination.7 Hence, a significant focus of current research is shifted to develop rapid and sensitive methods for the detection of foodborne pathogens. The efforts are directed to devise methods and systems that can be operated even by an untrained person. This is important to ensure more routine monitoring of the food quality and to prevent the infections and losses caused by contaminated food.

Biosensors are one of the most viable options as a point-of-care and portable solution for fast detection of bacteria with relative ease.8–10 During the last decade, nanomaterials have emerged as promising functional materials in biosensor fabrication.10–12 The use of nanomaterials in biosensor development enabled higher loading of biomolecules due to several meritful properties (e.g., high surface area).8,11,13 This ultimately facilitated the enhancement of sensitivity toward the bio-analytical assay. The nanomaterials also possess many other useful properties including small size, quantum confinement effects, high surface reactivity, and enhanced magnetic/electrical/optical properties, which can be tailored to optimize the desired specifications of the sensors. For achieving the desired specificity of biosensing applications, nanomaterials are generally conjugated with biomolecules, e.g. antibodies, enzymes, bacteriophages, nucleic acids, etc.14–17

Fluorescence based sensing is an excellent tool for detection of bacteria in food samples with advantages of high sensitivity, facile fabrication, and multiplexed detection.2,6,18,19 Several researchers have reported the development of fluorescent biosensors for S. aureus based on the application of nanomaterial–antibody/aptamer interfaces.20–22 For instance, an aptamer functionalized graphene oxide (GO) based biosensor was used in combination with a polydimethylsiloxane (PDMS)/paper hybrid to develop a microfluidic system for single-step detection of S. aureus.20 The fluorescence quenching property of GO was exploited for the assay development. In the presence of target bacteria (S. aureus), the fluorescently tagged aptamer molecules became detached from GO, resulting in the restoration of the fluorescence signal. The above developed assay demonstrated a limit of detection (LOD) of 8 × 102 CFU mL−1.20 He et al. (2014) proposed a dual color flow cytometric sensing platform using a combination of fluorescent silica nanoparticles (FSiNPs) and fluorescent dye (SYBR Green I).21 The FSiNP labelled aptamer was employed as a recognition probe while SYBER Green I was used to stain the nucleic acids. The above detection system for S. aureus offered LODs of 150 and 76 CFU mL−1 in buffer and milk samples, respectively.21 A similar sensing platform composed of aptamer conjugated fluorescent silica nanoparticles (Apts/FNPs) was developed for the detection of S. aureus in spiked water samples.22 The assay was based on positive dielectrophoresis (pDEP) driven on-line determination of S. aureus. The bacteria sample was incubated with Apts/FNPs prior to the injection into the microfluidic channel. An applied AC voltage resulted in the accumulation of Apts/FNP labelled S. aureus over the electrode gap owing to the pDEP force. The accumulated S. aureus were later detected using fluorescence microscopy imaging with detection limits of 9.3 × 101 and 2.7 × 102 CFU mL−1 in deionized and spiked water samples, respectively.22

Due to their diverse structural configurations and exciting optical properties, the metal–organic frameworks (MOFs) have attracted immense attention for biosensing applications.14,23,24 MOFs are hybrid materials consisting of metal ions linked to the organic ligands through coordination bonds to form 1-, 2-, or 3-D network structures. They are supramolecular assemblies with tuneable properties and functionality depending on the type of metal or ligand used during synthesis. Recently, luminescent MOFs have been proposed for several biological applications, including drug delivery, imaging, and molecular sensing.23–28 The luminescent MOFs show a number of peculiar advantages over other fluorescent molecules, e.g. crystallinity, nano-to-micron sized structures, stable fluorescence (with respect to time and temperature), and readily available functional groups for the conjugation of biorecognition species.29–31

For the first time, the present work reports the use of an optical (luminescent) biosensor, employing a non-toxic, biocompatible, and water-stable MOF, i.e., NH2-MIL-53 (Fe) as a fluorescent marker. To this end, this MOF has been interfaced with an anti-S. aureus antibody and the resulting biosensor has been investigated for the detection of S. aureus in different samples, including some real (spiked) samples. The specific binding of the bacteria with the bioprobe (i.e., antibody conjugated MOF) caused a reduction in the fluorescence intensity in proportion to the amount of bacteria added. This is attributable to the competitive absorption of the excitation energy by both the bioprobe and the attached bacteria (Graphical abstract). Hence, upon capturing the bacteria, the fluorescent bioprobe experienced a reduction in excitation energy which subsequently resulted in a decreased emission intensity. As our results have highlighted, the application of this new Ab/NH2-MIL-53 biosensor has allowed the sensitive detection of S. aureus over a wide concentration range at a considerably low limit of detection of 85 CFU mL−1. The results of this sensing technique, when applied for the detection of S. aureus in spiked water and cream pastry samples, agreed well with those of the standard colony counting method.

2. Experimental

2.1. Materials

A list of reagents used in the study include FeCl3·6H2O (ferric chloride, Sigma), NH2-BDC (2-aminobenzene-1,4-dicarboxylic acid; Sigma), EDC ((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), Sigma), NHS (N-hydroxysuccinimide, Sigma), Soya Casein Digest agar (SCD, Himedia), SCD broth (Himedia), and phosphate buffered saline (PBS) buffer (Himedia).

2.2. Equipment

For the characterization of materials used in this study, analysis was carried out using a number of instruments including a UV-Vis spectrophotometer (UV-Vis-NIR, Varian Cary 5000; scan rate: 600 nm min−1), Fourier-transform infrared spectrophotometer (FTIR, Nicolet, iS10; scan rate: 2.5 cm s−1), photoluminescence spectrophotometer (PL, Varian Cary), field-emission scanning electron microscope (FESEM, Hitachi S4300 SE/N), transmission electron microscope (TEM, TECNAI G2F-20), and X-ray diffractometer (XRD, Bruker, D8 Advance; Kα: 1.54 Å, scan speed: 4° min−1).

2.3. Synthesis of NH2-MIL-53 (Fe)

NH2-MIL-53 was prepared by a solvothermal method as reported elsewhere.32 Briefly, an equimolar mixture of FeCl3·6H2O (5 mmol) and NH2-BDC (5 mmol) was prepared in deionized (DI) water. It was then transferred to a sealed Teflon bottle and treated with autoclave heating at 150 °C for 3 days. The formed product (NH2-MIL-53) was collected by filtration and then washed twice with water and ethanol. Finally, the product was dried at 70 °C.

2.4. Bio-conjugation of anti-S. aureus antibodies with NH2-MIL-53

The antibody/NH2-MIL-53 complex (Ab/NH2-MIL-53) was prepared by conjugating the antibodies with the MOF as per the standard carbodiimide chemistry. Briefly, the amine functional group containing NH2-MIL-53 (2 mg mL−1) was dispersed in 10 mL of PBS buffer. Next, the antibody solution (0.1 mg mL−1 in 0.1 M PBS, which also contained 10 mM EDC and 5 mM NHS) was added. The resulting mixture was incubated at 4 °C overnight so that the carboxyl groups present on the Fc portion of the anti-S. aureus antibody conjugated with the pendant amine groups of the MOF via the formation of an amide linkage. The above formed Ab/NH2-MIL-53 complex was collected by centrifugation at 3214g for 10 min (4 °C). It was subsequently washed with PBS buffer (three times) to remove any unbound Ab or MOF particles. The purified Ab/NH2-MIL-53 was then stored under refrigerated conditions (4 °C).

2.5. Analysis of real samples

A demonstration of the practical utility of the Ab/NH2-MIL-53 sensory conjugate was carried out by the analysis of S. aureus in spiked samples of river water and cream pastry. For preparing the spiked river water sample, it was mixed with an unknown concentration of the bacteria. The sample was then filtered with a 0.22 μm hydrophilic filter paper. The bacteria separated on the filter paper were re-suspended in a similar volume of PBS buffer. In the case of cream pastry, 20 g of a pastry sample was transferred aseptically into a sterile plastic bag. A solvent mixture of 0.1% w/v peptone, 0.085% w/v NaCl, and 0.1% v/v Tween 80 was then used to homogenize the sample. Further dilutions were made in sterile peptone water (0.1%).

The validation of the data was carried out against the standard colony counting method. The samples with spiked bacteria were diluted in different fractions (e.g. 1×, 102×, 103×, and 104×) and then cultured to form colonies. A plate with easily countable number of colonies was analyzed to determine the bacterial concentration in all the prepared samples.

3. Results and discussion

3.1. Spectroscopic and structural characterization of Ab/NH2-MIL-53 bioconjugate

The confirmation about the successful formation of the Ab/NH2-MIL-53 bioprobe was done with several spectroscopic and structural studies. In UV-Vis analysis, NH2-MIL-53 exhibited absorption peaks at 220 and 340 nm which correspond to the –OH group sharing between FeO6 octahedral clusters bridged with ligand molecules.33 The presence of a protein specific peak around 280 nm indicated the successful linkage between the antibodies and NH2-MIL-53 (Fig. 1). FTIR investigations also showed the presence of bands around 3100–3500 (amine groups of NH2-MIL-53) and 1600–1700 cm−1 to signify the amide bond formation between the antibody and NH2-MIL-53 molecules33 (Fig. 2).
image file: c8ay02476f-f1.tif
Fig. 1 UV-Vis absorption spectra of NH2-MIL-53 and Ab/NH2-MIL-53.

image file: c8ay02476f-f2.tif
Fig. 2 FTIR spectra of NH2-MIL-53 and Ab/NH2-MIL-53.

PXRD patterns of both NH2-MIL-53 and Ab/NH2-MIL-53 are shown in Fig. 3. PXRD of NH2-MIL-53 was characterized with all the typical peaks matching well with the established structural data.32 As it is highlighted, the intrinsic crystallinity of the MOF was preserved even after the bio-conjugation with antibodies. Further, the FE-SEM investigations (Fig. 4) also verified that the Ab/NH2-MIL-53 complex inherited the primary structural properties of the parent fluorescent MOF.

image file: c8ay02476f-f3.tif
Fig. 3 XRD patterns of NH2-MIL-53 and Ab/NH2-MIL-53.

image file: c8ay02476f-f4.tif
Fig. 4 FE-SEM images of (a) NH2-MIL-53; (b) Ab/NH2-MIL-53.

3.2. Optical response of the Ab/NH2-MIL-53 bioprobe against various concentrations of bacteria

The photoluminescence (PL) response of the developed Ab/NH2-MIL-53 probe against varying concentrations of S. aureus (40–4 × 108 CFU mL−1) is depicted in Fig. 5. The sensor response was recorded after incubating a 400 μL sample of Ab/NH2-MIL-53 (1 mg mL−1) with 600 μL of 40 – 4 × 108 CFU mL−1 of the S. aureus solution (in PBS) for 20 min at room temperature (RT, 25 ± 2 °C). After the incubation period, the bacteria/Ab/NH2-MIL-53 complex was separated from the reaction mixture by centrifugation at 5000 rpm for 10 min. The collected residue (bacteria/Ab/NH2-MIL-53) was re-suspended in 1 mL of PBS buffer followed by the estimation of its PL intensity (excitation wavelength = 300 nm, emission peak wavelength = 430 nm).
image file: c8ay02476f-f5.tif
Fig. 5 (a) PL spectra of the Ab/NH2-MIL-53 complex against 40–4 × 108 CFU mL−1 of S. aureus; (b) corresponding calibration curve.

The Ab/NH2-MIL-53 bioprobe has allowed the detection of S. aureus by showing a concentration dependent quenching in the PL intensity (Fig. 5(a)). This observed phenomenon in quenching can be attributed to the attenuation in the effective excitation energy available to the Ab/NH2-MIL-53 probe after the bacteria–antibody binding. In other words, it may also reflect the results of the competitive absorption of light between the Ab/NH2-MIL-53 bioprobe and the captured bacteria. The calibration curve for the above sensor response is plotted and is shown in Fig. 5(b). Clearly, the Ab/NH2-MIL-53 bioprobe was capable of providing a logarithmically linear sensor response over a wide range (4 × 102 to 4 × 108 CFU mL−1) of analyte concentrations. The limit of detection (LOD) was calculated with the formula of 3σ/S (where σ and S corresponded to the standard deviation estimated for a blank sample and the slope of a calibration curve, respectively). Based on σ and S values of 3.5 and 0.1235, respectively, the LOD of the present Ab/NH2-MIL-53 based luminescent biosensing method was calculated to be 85 CFU mL−1. It is worth noting that the above value of LOD obtained with the Ab/NH2-MIL-53 system is better than or comparable with that of most of the earlier reported optical biosensors for S. aureus (Table 1).

Table 1 Comparative data of the sensing performance of fluorescent biosensors reported in the literature for the detection of S. aureus
Order Sensing material Important sensor characteristics Ref.
Range (CFU mL−1) LOD (CFU mL−1)
1 Ab/Quantum dot 103–105 103 18
2 (GO/PDMS)/paper 104–106 8 × 102 20
3 FSiNPs/SYBR Green I 3.4 × 102–3.4 × 104 (buffer) 1.5 × 102 (buffer) 21
6 × 102–6 × 105 (milk) 7.6 × 102 (milk)
4 Aptamers/FNPs 5 × 101–1 × 106 (deionized water) 9.3 × 101 (deionized water) 22
7 × 101–7 × 106 (spiked water) 2.7 × 102 (spiked water)
5 Ab/NH 2 -MIL-53 40–4 × 10 8 85 Present work

The specificity of the present sensor toward the target analyte (S. aureus, 4 × 102 CFU mL−1) in the presence of other possible interfering bacteria (E. coli and S. arlettae, 4 × 106 CFU mL−1) has also been tested. The results of this study are shown in Fig. 6(a). Due to the specific nature of the antibodies used, the Ab/NH2-MIL-53 bioprobe could very well tolerate the potential interferences of other bacteria as tested by above-mentioned ones.

image file: c8ay02476f-f6.tif
Fig. 6 (a) Specificity testing of Ab/NH2-MIL-53 towards S. aureus (4 × 102 CFU mL−1) in the presence of S. arlettae and E. coli (4 × 106 CFU mL−1) and (b) effect of the incubation time for the Ab/NH2-MIL-53 bioprobe to provide a stable response toward S. aureus (4 × 104 CFU mL−1).

The spiked samples of river water and cream pastry have also been analyzed with the Ab/NH2-MIL-53 bioprobe. The results of the PL based analysis along with those obtained with the standard colony counting method are given in Table 2. The performance and accuracy of the Ab/NH2-MIL-53 bioprobe matched nicely with the data of the standard colony counting method.

Table 2 Analysis of S. aureus in different (diluted) samples of spiked pastry cream: tests between the standard colony counting method and biosensing based on Ab/NH2-MIL-53a
Known concentration of S. aureus taken in the aqueous sample (CFU mL−1) Concentration of S. aureus as detected by different methods
Standard colony counting method (CFU mL−1) Ab/NH2-MIL-53 (CFU mL−1)
a Calculations for the colony counting method: CFU/mL = (number of colonies appeared on the plate × 10 × reciprocal of counted dilution)/(vol. of bacterial suspension).
4 × 102 3.9 × 102 3.9 (±0.03) × 102
4 × 104 3.9 × 104 3.9 (±0.02) × 104
4 × 106 3.9 × 106 4.0 (±0.05) × 106
4 × 108 3.9 × 108 4.0 (±0.06) × 108

3.3. Response time, inter- and intra- precision assay, and long-term stability of the Ab/NH2-MIL-53 bioprobe

The response time of the Ab/NH2-MIL-53 bioprobe was optimised by mixing and incubating it with a fixed concentration of the target bacteria (i.e. S. aureus, 4 × 104 CFU mL−1) for different time intervals. As revealed from data of this study (Fig. 6(b)), an incubation time of 15 minutes was required for attaining the desired sensor response (i.e., maximum degree of PL quenching). In all other studies, we kept the incubation time constant at 20 minutes for recording the sensor response.

The reproducibility of the Ab/NH2-MIL-53 bioprobe was determined by carrying out intra- and inter- assay precision measurements (Table 3). For intra-assay precision, the response of the bioprobe was checked against a fixed concentration of S. aureus (4 × 104 CFU mL−1) for five replicate determinations. The value of the relative standard deviation (RSD) was computed to be 4.3%. The inter-assay precision was determined by measuring a fixed concentration of S. aureus (4 × 104 CFU mL−1) with four different batches of the bioprobe prepared under identical conditions. The analysis of S. aureus with the above prepared different sets of the bioprobe exhibited an RSD of 6.47%. Both the above intra-assay and inter-assay studies gave a fair idea about the reproducibility of the Ab/NH2-MIL-53 bioprobe.

Table 3 Intra- and inter- assay precision measurement analysis of the Ab/NH2-MIL-53 bioprobe towards a fixed concentration of S. aureus (4 × 104 CFU mL−1)
Order Sample name PL quenching (%) RSD (%)
Intra-assay precision measurements
1 Replicate -1 12.51 4.3
2 Replicate -2 13.15
3 Replicate -3 11.84
4 Replicate -4 12.93
5 Replicate -5 12.16
[thin space (1/6-em)]
Inter-assay precision measurements
1 Batch-1 12.51 6.3
2 Batch-2 11.47
3 Batch-3 13.36
4 Batch-4 12.72

The long-term stability of the sensing material was investigated by recording its PL response as a function of the storage period. The Ab/NH2-MIL-53 complex was stored at 4 °C and used for the analysis of S. aureus (4 × 104 CFU mL−1) at periodic intervals. As the data shown in Fig. 7 revealed, the Ab/NH2-MIL-53 bioprobe was able to provide a stable sensing response even when it was kept over 60 days.

image file: c8ay02476f-f7.tif
Fig. 7 Results showing the stability of the response attained from the stored Ab/NH2-MIL-53 complex (60 days) toward S. aureus (4 × 104 CFU mL−1).

4. Conclusions

The present study has highlighted the potential of a luminescent bioprobe developed through bio-conjugation of the NH2-MIL-53 MOF and antibodies for simple yet highly efficient biosensing of S. aureus. The application of NH2-MIL-53 as a sensing material for the biosensing of bacteria has been demonstrated as an attractive choice because of some of its crucial advantages over other luminescent nanomaterials, e.g. organic dyes and quantum dots. NH2-MIL-53 provides stable luminescence while it does not contain any hazardous elements. Unlike other nanomaterials, NH2-MIL-53 does not require post-synthetic modifications or capping of the secondary layer before the attachment of antibodies. When compared with the existing fluorescent immunosensors for S. aureus, the Ab/NH2-MIL-53 bioprobe has delivered better or comparable performance in terms of both range of analysis and limit of detection. As such, the system showed excellent sensitivity and specificity while being stable even after the prolonged storage. The biosensing strategy proposed herein with the application of the antibody and MOF bioconjugate can be extended further for the design of bioprobing systems for many other forms of bacteria through the integration of suitable antibodies/aptamers/other recognition elements with MOFs.

Conflicts of interestThere are no conflicts to declare.


Authors are thankful to the Director, CSIR-CSIO, Chandigarh, for providing the necessary infrastructural facilities. Financial assistance to the work from the DBT (Department of Biotechnology, India) project grant BT/PR18868/BCE/8/1370/2016 is gratefully acknowledged. KHK acknowledges the support made by the R&D Center for Green Patrol Technologies through the R&D for Global Top Environmental Technologies funded by the Ministry of Environment (MOE) as well as by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant No. 2016R1E1A1A01940995).


  1. D. G. Newell, M. Koopmans, L. Verhoef, E. Duizer, A. Aidara-Kane, H. Sprong, M. Opsteegh, M. Langelaar, J. Threfall and F. Scheutz, Int. J. Food Microbiol., 2010, 139, S3–S15 CrossRef PubMed.
  2. J. W.-F. Law, N.-S. Ab Mutalib, K.-G. Chan and L.-H. Lee, Front. Microbiol., 2015, 5, 770 Search PubMed.
  3. N. Bhardwaj, S. K. Bhardwaj, M. K. Nayak, J. Mehta, K.-H. Kim and A. Deep, TrAC, Trends Anal. Chem., 2017, 97, 120–135 CrossRef CAS.
  4. Å. Rosengren, A. Fabricius, B. Guss, S. Sylvén and R. Lindqvist, Int. J. Food Microbiol., 2010, 144, 263–269 CrossRef PubMed.
  5. E. Larkin, R. Carman, T. Krakauer and B. Stiles, Curr. Med. Chem., 2009, 16, 4003–4019 CrossRef CAS PubMed.
  6. P. Mandal, A. Biswas, K. Choi and U. Pal, Am. J. Food Technol., 2011, 6, 87–102 CrossRef.
  7. P. D. Skottrup, M. Nicolaisen and A. F. Justesen, Biosens. Bioelectron., 2008, 24, 339–348 CrossRef CAS PubMed.
  8. J. Wang, Biosens. Bioelectron., 2006, 21, 1887–1892 CrossRef CAS PubMed.
  9. D. Zhang and Q. Liu, Biosens. Bioelectron., 2016, 75, 273–284 CrossRef CAS PubMed.
  10. J. Breault-Turcot and J.-F. Masson, Anal. Bioanal. Chem., 2012, 403, 1477–1484 CrossRef CAS PubMed.
  11. N. Sanvicens, C. Pastells, N. Pascual and M.-P. Marco, TrAC, Trends Anal. Chem., 2009, 28, 1243–1252 CrossRef CAS.
  12. M. M. Barsan and C. M. Brett, TrAC, Trends Anal. Chem., 2016, 79, 286–296 CrossRef CAS.
  13. J. Wang, Analyst, 2005, 130, 421–426 RSC.
  14. N. Bhardwaj, S. K. Bhardwaj, J. Mehta, M. K. Nayak and A. Deep, New J. Chem., 2016, 40, 8068–8073 RSC.
  15. G. Luka, A. Ahmadi, H. Najjaran, E. Alocilja, M. DeRosa, K. Wolthers, A. Malki, H. Aziz, A. Althani and M. Hoorfar, Sensors, 2015, 15, 30011–30031 CrossRef CAS PubMed.
  16. Y. Du and S. Dong, Anal. Chem., 2016, 89, 189–215 CrossRef PubMed.
  17. S. Sharma, H. Byrne and R. J. O'Kennedy, Essays Biochem., 2016, 60, 9–18 CrossRef PubMed.
  18. Y. Zhao, M. Ye, Q. Chao, N. Jia, Y. Ge and H. Shen, J. Agric. Food Chem., 2008, 57, 517–524 CrossRef PubMed.
  19. H. Fukushima, Y. Tsunomori and R. Seki, J. Clin. Microbiol., 2003, 41, 5134–5146 CrossRef CAS.
  20. P. Zuo, X. Li, D. C. Dominguez and B.-C. Ye, Lab Chip, 2013, 13, 3921–3928 RSC.
  21. X. He, Y. Li, D. He, K. Wang, J. Shangguan and H. Shi, J. Biomed. Nanotechnol., 2014, 10, 1359–1368 CrossRef CAS.
  22. J. Shangguan, Y. Li, D. He, X. He, K. Wang, Z. Zou and H. Shi, Analyst, 2015, 140, 4489–4497 RSC.
  23. S. K. Bhardwaj, N. Bhardwaj, G. C. Mohanta, P. Kumar, A. L. Sharma, K.-H. Kim and A. Deep, ACS Appl. Mater. Interfaces, 2015, 7, 26124–26130 CrossRef CAS PubMed.
  24. O. Shekhah, J. Liu, R. Fischer and C. Wöll, Chem. Soc. Rev., 2011, 40, 1081–1106 RSC.
  25. S. K. Bhardwaj, G. C. Mohanta, A. L. Sharma, K.-H. Kim and A. Deep, Anal. Chim. Acta, 2018, 1043, 89–97 CrossRef CAS PubMed.
  26. N. Bhardwaj, S. Bhardwaj, J. Mehta, K.-H. Kim and A. Deep, Biosens. Bioelectron., 2016, 86, 799–804 CrossRef CAS PubMed.
  27. S. K. Bhardwaj, A. L. Sharma, N. Bhardwaj, M. Kukkar, A. A. Gill, K.-H. Kim and A. Deep, Sens. Actuators, B, 2017, 240, 10–17 CrossRef CAS.
  28. P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, T. Baati, J. F. Eubank, D. Heurtaux, P. Clayette and C. Kreuz, Nat. Mater., 2010, 9, 172 CrossRef CAS PubMed.
  29. S. M. Cohen, Chem. Sci., 2010, 1, 32–36 RSC.
  30. J.-N. Hao and B. Yan, Chem. Commun., 2015, 51, 7737–7740 RSC.
  31. B. Chen, S. Xiang and G. Qian, Acc. Chem. Res., 2010, 43, 1115–1124 CrossRef CAS PubMed.
  32. D. Wang, R. Huang, W. Liu, D. Sun and Z. Li, ACS Catal., 2014, 4, 4254–4260 CrossRef CAS.
  33. N. Bhardwaj, S. K. Bhardwaj, J. Mehta, K.-H. Kim and A. Deep, ACS Appl. Mater. Interfaces, 2017, 9, 33589–33598 CrossRef CAS PubMed.

This journal is © The Royal Society of Chemistry 2019