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Analytical Chemistry
Photoluminescent Lateral-Flow Immunoassay Revealed by Graphene Oxide: Highly Sensitive Paper-Based Pathogen Detection Eden Morales-Narváez∫, Tina Naghdi∫₰‡, Erhan Zor∫§‡, and Arben Merkoçi∫†*. ∫ ICN2 – Nanobioelectronics & Biosensors Group, Institut Catala de Nanociencia i Nanotecnolo-gia, Campus UAB, Bellaterra (Barcelona) 08193, Spain ₰ Department of Chemistry, College of Science, Shahid Chamran University, Ahvaz, Iran § Selcuk University, Education Faculty, Konya 42090, Turkey † ICREA − Institucio Catalana de Recerca i Estudis Avançats, Barcelona 08010, Spain ABSTRACT: A paper-based lateral flow immunoassay for pathogen detection that avoids the use of secondary antibodies and is revealed by the photoluminescence quenching ability of graphene oxide is reported. Escherichia coli has been selected as a model pathogen. The proposed device is able to display a highly specific and sensitive performance with a limit of detection of 10 CFU mL-1 in standard buffer and 100 CFU mL-1 in bottled water and milk. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications.
Foodborne and waterborne infections result from ingesting food products or drinks contaminated by pathogens. Being a serious threat, foodborne and waterborne illnesses often lead to a considerable number of deaths and substantial deficits in productivity.1 In fact, according to The U.S. Department of Agriculture’s Economic Research Service, the cost of foodborne-illness may range from 14.1 to 152 billion dollars.2 In addition, pathogens have always evolved and they can resist the new drugs that are used to combat them. In fact, drug resistant related infections are expected to be more deadly diseases than cancer by 2050 (they will kill an extra 10 million people a year worldwide) unless action is taken.3 Thus, early detection of disease-causing bacteria is crucial to preserve food and beverage safety and safeguard public health. Conventional bacteria detection methods such as the plating technique and biochemical methods are reliable, highly robust and accurate. However, they are not particularly suitable to be used as user-friendly approaches since they require culture processes, trained operators, relatively sophisticated laboratory equipment and time-consuming labors (e.g. up to 3 days).4 Presently, the development of new tools that are fast, highly sensitive, disposable, cost effective, easy-to-use and amenable to portability and automation is under active research. Due to the unique structural, electronic and optical properties offered by nanomaterials, nanotechnological approaches feature the majority of such developments.5,6 Due to their virtually universal, long-range and highly efficient photoluminescence quenching capabilities, graphenerelated materials are being widely assessed for biosensing applications based on energy transfer.7–9 On the other hand, quantum dots nanocrystals (QDs) exhibit distinctive photophysical properties (such as size-tunable emission, narrow and symmetric photoluminescence, broad and strong excitation spectra, strong luminescence and robust photostability) that
have been intensely applied in advantageous immunoassays.10– 13 Previously, our research group has explored the photoluminescence quenching ability and the oxygenated twodimensional network of graphene oxide (GO) in order to exploit it as a pathogen-revealing agent while using a conventional glass slide-based microarray system, which is expensive and not particularly appealing for portability.14,15 Herein, taking advantage of the mentioned research, a simple, versatile and highly sensitive photoluminescent lateral flow immunoassay for pathogen detection is reported. The proposed biosensing approach consist of a paper-based lateral flow immunoassay, which is an integrated technique enabling a detection device in a simple strip (made of nitro/cellulose). Typically, the sample flow is driven by capillary forces throughout different sections of the strip containing the necessary reagents for different roles.16 This novel lateral flow approach bears a test line with antibody decorated CdSe@ZnS quantum dots (Ab-QDs) printed as photoluminescent probes and a control line made of bare QDs. After applying the sample to the lateral flow strip, GO is added as a revealing-agent. When the analyzed sample does not contain the target pathogen, the test line is efficiently quenched by resonance energy transfer since the distance between the Ab-QDs (donors) and GO (acceptor) is around few nanometers resulting in an ‘OFF state’ of the test line.17,18 In contrast, when the analyzed sample contains the target, the pathogen is specifically recognized and linked/attached by the Ab-QDs of the test line and, after adding GO, resonance energy transfer is hindered as the distance between donor and acceptor exceeds the distance in which resonance energy transfer is observable (>20 nm)15,17,18 leading to an ‘ON state’ of the test line. Likewise, since the control line has no pathogen-binding biomolecules (i.e. antibodies), it is always quenched to ensure that the assay was carried out correctly, see Figure 1. Conventional lateral flow
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immunoassays rely on labelling processes that mainly occur in both, the test and control line.16 However, the proposed immunoassay only requires antibodies onto the test line and lacks any immunolabelling detection/control process as usually reported so far for QDs19 or gold nanoparticles20–22 based lateral flow devices.23
Figure 1. Photoluminescent lateral flow test revealed by graphene oxide (GO) for pathogen detection. A. Schematic representation. Quantum dots (QDs) are printed on nitrocellulose substrate as control line. Antibody-decorated quantum dots are printed on the same substrate as test line. The sample flows from the sample pad (SP) to the absorbent pad (AP). Upon excitation, the control line is always quenched by a GO flow (since the distance between QDs energy transfer donors and GO acceptor is minimum). The test line is quenched by a graphene oxide flow (GO) when the analyte is not present. The contrary, when the analyte is present it is selectively captured and the test line will not be significantly quenched when compared with the control line (since the distance between donor and acceptor exceeds the nanoscale due to the analyte size). B. Portable lateral flow reader and typical positive /
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negative response of the test. The graph contains the profile of the original intensity of the strip (dotted line), the final intensity of a positive assay (red line) and the final intensity of a negative assay (green line).
EXPERIMENTAL SECTION Reagents and Equipment. All commercial reagents were of analytical grade and handled according to the material safety data sheets suggested by the suppliers. Graphene oxide (GO) was purchased from Angstron Materials (Dayton, OH, USA). Anti-E.coli antibody (Ab, ab68451) was obtained from Abcam (Cambridge, UK) and Streptavidin–Quantum dot 655 was from Life Technologies (Carlsbad, CA, USA). Phosphate buffered saline tablet (P4417), bovine serum albumin (BSA), Tween-20 were purchased from Sigma-Aldrich (Madrid, Spain). The laminated cards (HF000MC100), nitrocellulose membrane (SHF1800425), sample and absorbent pads (CFSP001700) for the production of the lateral flow assay (LFA) strips were purchased from Millipore (Billerica, MA, USA). Escherichia coli O157:H7 (CECT 4783, E. coli) and Salmonella typhimurium (CECT 722T, S. typhimurium) strains were obtained from the Colección Española de Cultivos Tipo (CECT, Valencia, Spain). PBS (10 mM, pH 7.4) supplemented with Tween 20 at 0.05% (v/v) (PBST) was used as washing buffer solution. PBS (10 mM, pH 7.4) with 0.5% (v/v) Tween 20 containing 1% of BSA fraction V (w/v) was employed as standard buffer. All aqueous solutions were freshly prepared in Milli-Q water produced using a Milli-Q system (>18.2 MΩ cm−1) purchased from Millipore. An IsoFlow reagent dispensing system (Imagene Technology, Hanover, NH, USA) was used to dispense the detection and control lines. A Dahle 533 guillotine (Dahle, Peterborough, NH, USA) was used to cut the strips for the desired size (6 mm). TS-100 Thermo-Shaker (Biosan, Riga, Latvia) was used as the stirrer for modification of QDs with antibodies. An ESEQuant lateral flow reader was performed to analyze the strips (Qiagen GmbH, Stockach Germany). A JP Selecta 2000210 oven (JP Selecta s.a., Barcelona, Spain) was used for drying the strips. SEM imaging was performed through a Magellan 400L SEM High Resolution SEM (FEI, Hillsboro, OR, USA). QDs conjugation with Antibodies (Ab). The stock suspension of Streptavidin-QDs and biotinylated antibodies were 1000 nM and 4.5 mg mL-1, respectively. QDs were mixed with Ab in standard buffer at a final concentration of 1.5 nM and 100 µg mL-1, respectively. The mixing of this solution was carried out at 650 rpm, at 4 ºC for 30 min. Preparation of the Strips. The fabrication of the explored test strips is based on four major steps: i) Assembly of the detection pad onto the laminated card. ii) Dispensing the test and control lines onto the detection pad (cellulose membrane) using IsoFlow reagent dispensing system. For this step, 1.5 nM / 100 µg mL−1 of Streptavidin–Quantum dot 655 / Ab and 1.5 nM of Quantum dot 655 suspensions were used to print the test and control lines, respectively. Then, the membrane was stored in the fridge at 4 °C by covering it with aluminum foil for overnight. The detection pad (of 2.5 x ~7 cm) was pretreated by adding 5 mL of standard buffer homogeneously and stored at ~4 ºC for 15 min and dried at ~37 °C for 2 h before the fourth step. iii) The preparation of the sample and absorbent pads by respectively washing with freshly prepared MilliQ water, PBST and standard buffer and drying in oven at ~37 °C for overnight. iv) Assembly of the sample and absorbent
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Analytical Chemistry
pads onto the prepared membrane (laminated card) and cutting the strips 6 mm wide prior to use. Lateral Flow Assay for Pathogen Detection: Overall procedure. E. Coli and S. typhimurium strains were prepared as previously described.15 E. Coli or S. typhimurium samples were prepared at different concentrations (e.g. 2, 5, 10, 102, 101, 105, 107 CFU mL-1) in standard buffer, bottled water or milk samples and stored in the fridge at 4 °C. Firstly, the initial photoluminescence intensity of the control and test lines of the lateral flow strips was obtained using the aforementioned lateral flow reader and 100 µl of the analyte suspensions were dispensed onto the sample pad of the fabricated test strips. After drying the strips, they were washed twice with 100 µl of PBST by adding this washing solution onto the sample pad (in order to remove any possible interference) and were dried in oven at ~37 °C for 1.5 h. Momentary, extra absorbent pads may be added on top of the original absorbent pads to assist this washing process. Secondly, 100 µl of 90 µg mL-1 of water-based graphene oxide (GO) dispersion containing 0.05% Tween 20 was dispensed onto the sample pad of the strips. Since QDs exhibit better photoluminescent performance in solid phase than in liquid phase,24 the strips were dried in oven at ~37 °C for 1.5 h before reading step. After drying, the final photoluminescence intensity of the control and test lines of the lateral flow strips was obtained using the aforementioned lateral flow reader.
mension of ~ 500 nm and showing a C/O ratio around 1 unit (manufacturer’s data, approximate values) was utilized in this research. Figure S1 displays a picture of the employed waterbased GO suspension, which is highly stable due to its oxygen-containing moieties and oxidation grade. An ESEQuant portable lateral flow reader that bears both, a 365 nm excitation source and an emission filter around 670 nm has been employed to measure the photoluminescence of the lateral flow strips, see Figure 1B. Herein, Escherichia coli (E. coli) has been employed as a model pathogen. The concentration of QDs printed on the test and control line was selected so as to bear c.a. 75% of the dynamic range of the lateral flow reader and avoid its saturation. This optimal QDs concentration was found to be 1.5 nM, see Figure S2 included as supporting information (SI). QDs were conjugated with anti-E. coli antibodies (Ab) as detailed in the Experimental Section. The optimum GO concentration was determined to be 90 µg mL-1, since it triggers a maximum quenching efficiency around 80% (i.e. 20% of the test/control line initial intensity is observed after applying GO), whereas lower concentrations bear less quenching efficiency and higher concentrations give rise to a blockage of the GO flow, affecting the quenching efficiency, see Figure S3. Consequently, [QDs] ≈1.5 nM and [GO] ≈ 90 µg mL-1 were employed throughout this biosensing approach. It is worth mentioning that this is the optimal concentration while using GO displaying the aforementioned characteristics. Hence, using a different kind of graphene-based material (i.e. with different average lateral size, number of layers or oxidation grade) would require a new optimization process. GO is likely to interact with the control and detection lines by electrostatic interactions.[8,14] Scanning-electron micrographs of the biosensing platform are shown in Figure 2.
Figure 2. Scanning-electron micrographs of the paper-based biosensing platform. A-B. Bare detection line. C-D. Graphene oxidecoated detection line.
RESULTS AND DISCUSSION Cellulose membrane was used as sample and absorbent pad (in other words, the initial and final sections of the strip, which are in charge of loading and pumping the sample respectively). Nitrocellulose membrane was used as detection pad. In accordance with previous characterizations,25 streptavidinfunctionalized CdSe@ZnS QDs donors that display a rice-like shape, an average size around 14±2 nm and maximum emission wavelength at ~665 nm were used throughout these experiments. In order to enforce reproducibility and repeatability of graphene-based technologies, graphene-based materials are suggested to be classified according to their number of layers, lateral size, and C/O ratio.26 Herein, a water-based dispersion of single layer GO microsheets acceptors with lateral dimensions ranging from 0.18 to 1.2 µm, average lateral (x-y) di-
Figure 3. Response of the photoluminescent lateral flow immunoassay revealed by graphene oxide (GO). A-B. E. coli detection in standard buffer. A. Bars representing the quenching of the test line (QTL, in orange) and the quenching of the control line (QCL, in blue) as a function of E. coli concentration (obtained by dividing the original intensity into the final intensity in the explored line). B. Normalization of the tests in A (QTL / QCL.). C-D. Selectivity and specificity assessment. C. Behaviour of the test in the presence of i) a high concentration of non-target pathogen (S. typhimurium), ii) a mix of high concentrations of both, S. typhimurium and E. coli, iii) a mix of a high concentration of S. typhimurium a and a low concentration of E. coli. D. Normalization of the tests in C. The threshold in green (B,D) represents the limit of detection
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of the proposed device (which is obtained as the mean value of the blank solutions plus three times their standard deviation). Error bars were obtained by taking the standard deviation of at least three strips assayed in the same conditions. The error bars represent the standard deviation of at least 3 tests.
The overall performance of the proposed lateral flow test was explored varying the concentration of the analyte (from 2 to 105 CFU mL-1) in a standard buffer (phosphate buffered saline, 0.01 M, supplemented with BSA, 1% w/v, and Tween 20, 0.5% v/v). Blank solutions were also tested to estimate the maximum quenching levels of both the test and the control line. The photoluminescence quenching levels were estimated in dimensionless units by dividing the final photoluminescence intensity into the original photoluminescence intensity of the respective line, from now on termed as quenching of the test line (QTL) and quenching of the control line (QCL), where the values close to 1 indicate a minimum quenching and the values close to 0 indicate a maximum quenching. As expected, QTL was observed to be modulated upon addition of the analyte and GO sequentially, whereas QCL was observed to be relatively constant (around 0.2 units) upon addition of the analyte and GO sequentially, see Figure 3A. The result of each strip was normalized by using the QTL/QCL ratio, where the assayed blank solutions displayed a QTL/QCL ratio around 1 and the samples containing the analyte displayed a QTL/QCL ratio greater than 1. The mean value of the QTL/QCL ratio of the blank solutions analysis plus 3 times its standard deviation was proposed to determine a threshold for the limit of detection of this lateral flow test. Interestingly, although this novel lateral flow immunoassay only requires antibodies onto the test line and lacks any labelling process (no analyte labelling with a secondary antibody in the test line, neither antibodies in the control line are required), this paper-based test was observed to display a highly sensitive response since the limit of detection of the samples assayed in standard buffer is around 10 CFU mL-1, see Figure 3B.
Figure 4. Performance of the lateral flow test in spiked samples of bottled water (A) and milk (B). The error bars represent the standard deviation of at least 3 tests.
Aiming at evaluating the specificity and selectivity of the proposed lateral flow test, Salmonella typhimurium (S. typhimurium) has been used as a non-target pathogen. As displayed in Figure 3C, different tests were carried out in standard buffer as follow: i) using a high concentration of S. typhimurium (107 CFU mL-1), ii) using a mix sample of high concentrations of both, S. typhimurium and E. coli (both at 107 CFU mL-1) and iii) using a mix sample of a high concentration of S. typhimurium (107 CFU mL-1) and a low concentration of E. coli (102 CFU mL-1). The QTL/ QCL ratio of the test i) is not greater than the threshold proposed as limit of detection, whereas low and high concentrations of the target pathogen in the presence of high concentrations of non-target pathogen trigger a QTL/QCL ratio greater than the limit of de-
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tection. These experiments suggest that the proposed lateral flow test is highly specific and selective, see Figure 3D. In order to study the performance of the test in different matrices, several concentrations of the analyte were respectively spiked in bottled water and milk samples in order to be assayed with the proposed lateral flow strips. Blank solutions of both, bottled water and milk samples were also assayed. The photoluminescence of the studied test lines was also effectively modulated upon addition of the spiked samples and GO. After obtaining the respective QTL/QCL ratios, the analysis of blank solutions of bottled water presented a QTL/QCL ratio around 1.25, whereas blank solutions of milk displayed a QTL/QCL ratio around 1. The limit of detection of the test in the assayed bottled water and milk samples was observed to be around 100 CFU mL-1 (see Figure 4). These results suggest that the performance of this lateral flow test is influenced by the matrix of the sample, which is a common phenomenon in immunoassays.27 However, it can also be associated with microenvironment changes (polarity and hydrogen-bonding capability of the matrix and the local viscosity, pH, and ionic strength), which are likely to affect the QDs photoluminescent properties.28 Even so, the test also displays a very sensitive response. Importantly, the studied tests were carried out using different batches of fabrication respectively, which demonstrate the reproducibility of the reported fabrication process. As it can be observed in Figure 3 and 4, the operative range of the strips showed a coefficient of variation below 30%, particularly from: 3 to 12% in PBS samples, 6 to 12% in bottled water samples and 5 to 22% in milk samples, which potentially meets the criteria toward validation of immunoassays.29
CONCLUSION The performance of a label-free lateral flow immunoassay for pathogen detection that obviates the use of secondary antibodies and is revealed by the photoluminescent quenching ability of GO has been explored. The analyte is selectively attached onto the test line of the strip (which contains AbQDs) leading to a highly specific and sensitive sensing phenomenon that is clearly observable up to 10 CFU mL-1 in standard buffer and 100 CFU mL-1 in bottled water and milk. Since the estimated cost of the materials and reagents of the proposed lateral flow test is less than 0.10 Euro (see table ST1 in SI), this approach will prove valuable as a cost-effective device that is disposable and easy-to-use. Moreover, this device is appealing for portable and automated applications that may include other similar size analytes with interest for diagnostics.
ASSOCIATED CONTENT Supporting Information Optimization of quantum dots and graphene oxide concentration (Figure S1 and S2, respectively) and the estimated cost of the proposed lateral flow test (in terms of materials and reagents) are available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *
[email protected] Present Addresses
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Analytical Chemistry
∫ ICN2 – Nanobioelectronics & Biosensors Group, Institut Catala de Nanociencia i Nanotecnolo-
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Quesada-González, D.; Merkoçi, A. Biosens. Bioelectron. 2015, 73, 47–63.
gia, Campus UAB, Bellaterra (Barcelona) 08193, Spain ₰ Department of Chemistry, College of Science, Shahid Chamran University, Ahvaz, Iran
§ Selcuk University, Education Faculty, Konya 42090, Turkey † ICREA − Institucio Catalana de Recerca i Estudis Avançats, Barcelona 08010, Spain
Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.
ACKNOWLEDGMENT This work was supported by MINECO, (Spain, BIO2013-49464EXP). ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). T.N and E.Z contributed equally to this work.
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A label-free lateral flow immunoassay for pathogen detection is revealed by the photoluminescent quenching ability of graphene oxide. The target bacteria is selectively attached onto the test line of the strip, which contains quantum dots complexed with antibodies, leading to a highly specific and sensitive biosensing system. This cost-effective and user-friendly device is appealing for portable and automated applications.
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