Colloids and Surfaces B: Biointerfaces 132 (2015) 264–270

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Qualitative and quantitative detection of T7 bacteriophages using paper based sandwich ELISA Mohidus Samad Khan a,c,∗ , Tripti Pande b , Theo G.M. van de Ven a,∗∗ a b c

Department of Chemistry, McGill University, Montreal, Quebec H3A 2A7, Canada Department of Microbiology and Immunology, McGill University, Montreal, Quebec H9R5X3, Canada Department of Chemical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka 1000, Bangladesh

a r t i c l e

i n f o

Article history: Received 16 February 2015 Received in revised form 14 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Keywords: T7 bacteriophages Viruses Antibodies Sandwich ELISA Paper diagnostics

a b s t r a c t Viruses cause many infectious diseases and consequently epidemic health threats. Paper based diagnostics and filters can offer attractive options for detecting and deactivating pathogens. However, due to their infectious characteristics, virus detection using paper diagnostics is more challenging compared to the detection of bacteria, enzymes, DNA or antigens. The major objective of this study was to prepare reliable, degradable and low cost paper diagnostics to detect viruses, without using sophisticated optical or microfluidic analytical instruments. T7 bacteriophage was used as a model virus. A paper based sandwich ELISA technique was developed to detect and quantify the T7 phages in solution. The paper based sandwich ELISA detected T7 phage concentrations as low as 100 pfu/mL to as high as 109 pfu/mL. The compatibility of paper based sandwich ELISA with the conventional titre count was tested using T7 phage solutions of unknown concentrations. The paper based sandwich ELISA technique is faster and economical compared to the traditional detection techniques. Therefore, with proper calibration and right reagents, and by following the biosafety regulations, the paper based technique can be said to be compatible and economical to the sophisticated laboratory diagnostic techniques applied to detect pathogenic viruses and other microorganisms. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Different biotechnological and biomedical applications are commonly restricted by the high cost and limited availability of tests and materials. In the detection of health conditions through pathological diagnosis, the outsourcing of blood, urine and other biofluid samples to an analytical laboratory is a standard practice. Reliable instantaneous diagnoses without recourse to sophisticated laboratory and analytical instrumentation would be a significant innovation that promises to improve health outcomes in underprivileged and remote areas [1–4]. In addition, access to simple, dependable and low cost water and air filters that are able to capture and deactivate waterborne and airborne pathogens would be

∗ Corresponding author at: Department of Chemical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka 1000, Bangladesh. Tel.: +88 01552352461. ∗∗ Corresponding author. E-mail addresses: [email protected] (M.S. Khan), [email protected] (T.G.M. van de Ven). http://dx.doi.org/10.1016/j.colsurfb.2015.05.028 0927-7765/© 2015 Elsevier B.V. All rights reserved.

equally invaluable for preventing epidemic health threats through provision of safe water and air [4–6]. Paper based diagnostics and filters offer attractive options for detecting and deactivating pathogens. Bioassays made of disposable materials can be used for regular tests to detect blood typing, cancers, generic conditions, and epidemic diseases such as hepatitis and influenza [4,5,7–10]. They can also be used to identify and filter heavy metals, chemical compounds and microbial activities in water. Successful commercialization requires bioassays to be low cost, which is best achieved through a high volume manufacturing process and with available raw materials. Paper has the potential to meet these criteria. Researchers demonstrated that antibody and enzyme active papers can detect ABO blood typing [1], bacteria [11], DNA [12], alcohol content in the breath [13], seafood freshness [14], and pathogens in biofluids [15–20]. However, simplicity, sensitivity, and amount of biofluid volume required for the test are ongoing challenges for developing paper based devices to detect pathogens, such as viruses [17–21]. Every year throughout the world, millions of people get infected by different waterborne and airborne viral diseases, such as: hepatitis, SARS, AIDS, diarrhoea, polio, chickenpox, smallpox and influenza, which are transmitted through drinking water, sexual

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contact, blood, saliva, breast milk and other body fluids [22–28]. Right diagnosis and treatment of these diseases cost billions of dollars, and still several millions of the infected people die every year [22–25,27,28]. Right diagnosis and early detection of those diseases and corresponding viruses can prevent epidemic health threats in many developing countries. Viruses are small in size, often infectious and therefore, requires sophisticated higher level biosafety laboratory to work with. This study aims to develop paper bioassays to detect viruses. T7 bacteriophage was used as a model virus. T7 bacteriophages are not infectious to human, and therefore, experiments with T7 phages can be designed and conducted at biosafety level 1 laboratories [29]. T7 bacteriophages are lytic bacteriophages capable to infect bacterial cells such as Escherichia Coli. The other major advantage of using T7 phages is the availability of T7 specific primary and secondary antibodies [30,31]. In this study, a paper based sandwich ELISA technique was developed and demonstrated to detect and quantify the T7 phages in solution. Two monoclonal antibodies (IgM) specific to T7 phage, were used as the primary [30] and secondary antibodies [31]. The secondary antibody [31] was conjugated with horseradish peroxidase (HRP) enzymes to give a colorimetric indication in the presence of T7 phage in solution.

2. Paper based Sandwich ELISA ELISA, enzyme-linked immunosorbent assay, is an enzyme immunoassay technique which is used to detect and report the presence of antigens, antibodies, proteins and pathogens using enzyme linked antibodies. ELISA techniques involve several incubation steps to coat antibodies, antigens and/or blocking agents, followed by a series of washing steps. Protein coating steps may require an incubation period between four hours to overnight; hence, performing an ELISA test to detect an analyte may take from several hours to as long as a day [32]. Elisa techniques can be broadly classified as direct ELISA, indirect ELISA, and Sandwich ELISA [32,33]. In the sandwich ELISA technique the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody, and offers higher sensitivity than direct ELISA or indirect ELISA techniques [32–34]. In sandwich ELISA, a surface is prepared using a primary antibody specific to the target molecule. Once the target molecule is applied, the primary antibody interacts with the target molecule to form an antibody-target molecule complex. In order to detect and report the target molecule attached to the primary antibody, an enzyme conjugated secondary antibody specific to the molecule is added to the system. This secondary antibody binds the other end of the target molecule leading to a sandwich structure encompassing the primary antibody, target molecule and secondary antibody complex. The enzyme conjugated to the secondary antibody reports the presence of the target molecule by reacting with the enzyme substrate and forming a colour product. In the paper based sandwich ELISA technique, a paper surface was prepared with the primary antibody. T7 bacteriophage was used as the target molecule. Fig. 1 indicates different steps of paper based sandwich ELISA test to detect T7 bacteriophages. Anti-T7 tag monoclonal IgM antibodies (mouse IgM isotype), specific to T7 tag sequence MASMTGGQQMG-K, were used as the primary and secondary antibodies [30,31]; the secondary antibody was conjugated with horseradish peroxidase (HRP) and recognized the T7 tag sequence on T7 bacteriophage. The presence of the secondary antibody could be found from the colour product produced from the enzyme-substrate reaction. Horseradish peroxidase (HRP) is a single chain polypeptide enzyme which readily combines with hydrogen peroxide (H2 O2 ) and the resultant, HRPH2 O2 complex, can oxidize a wide variety of chromogenic hydrogen

Fig. 1. Schematic of paper based sandwich ELISA. (a) Antibody (primary) – T7 phage – antibody (secondary) complex formation on paper surface in presence of T7 phages; (b) no antibody-T7 phage-antibody complex forms in absence of T7 phage.

donors [35]. Most reactions catalyzed by HRP can be expressed by the following equation [36]: HRP

H2 O2 + 2AH2 −→2H2 O + 2AH•

(1)

and AH• represent the reducing substrate and its radical

where, AH2 product, respectively. 3,3 -Diaminobenzidine (DAB) is an organic compound that is used as a substrate for horseradish peroxidase (HRP) enzyme. In the presence of HRP, DAB is oxidized by hydrogen peroxide, producing a strong brownish colour complex that is stable. This can be observed visually and does not fade upon exposure to light. The HRP-DAB reaction can be expressed by the following equations [37]: HRP

H2 O2 + DAB−→insoluble, brown-black precipitate

(2)

Fig. 1a indicates the two layers of anti-T7 tagged antibodies which form the antibody-virus-antibody sandwich complex, in the presence of a T7 bacteriophage. The HRP conjugated with secondary antibody reacts with the enzyme substrate. The colour product formed on the paper surface from the enzyme-substrate reaction reports the presence of T7 phage in the system. In the absence of T7 phages, the primary and secondary antibodies do not bind with each other and consequently do not form a sandwich complex. 3. Experimental 3.1. Materials T7 bacteriophages were cultured in the laboratory using Escherichia coli ATTC® BAA-1025 (American Type Culture Collection (ATCC), Manassas, USA) as the host and Escherichia coli bacteriophage T7 ATTC® BAA-1025-B2 (American Type Culture Collection (ATCC), Manassas, USA) as the seed. LB broth (Sigma L3152; (Sigma–Aldrich, Missouri, USA) and agar (Fluka 05039;

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Fluka, Taufkirchen, Germany) were used as cultural medium and to prepare agar plates. Two anti-T7 tag monoclonal IgM antibodies (mouse IgM isotype): abcam ab50545 (Abcam plc, Cambridge, UK) and Sigma T3699 (Sigma–Aldrich, Missouri, USA), specific to T7 tag sequence MASMTGGQQMG-K, were used as the primary and secondary antibodies, respectively. Primary antibody solution (2 ␮g/mL) (abcam ab50545) was deposited on filter paper (Whatman#42; particle retention diameter 2.5 ␮m). The secondary antibody was conjugated with horseradish peroxidase (HRP) and recognized the T7 tag sequence on T7 bacteriophage. The presence of secondary antibody can be visualized via the colour product produced from the enzyme-substrate reaction. A solution of the secondary antibody (Sigma T3699) of 20 ␮g/mL concentration was prepared to ensure a strong colour product formation from the enzyme-substrate reaction. The HRP activity of the 20 ␮g/mL secondary antibody (Sigma T36990 solution was 0.5 U/mL. 1% solution of bovine serum albumin (BSA) (Sigma A9576; Sigma–Aldrich, Missouri, USA) was used to block the nonreactive surface area of filter paper. Diaminobenzidine (DAB) substrate (Sigma D4168; Sigma–Aldrich, Missouri, USA), containing 3,3 -diaminobenzidine (DAB) and urea hydrogen peroxide, was used to react with the HRP enzyme conjugated with secondary antibodies. Phosphate buffer saline (0.01 M) (PBS; Sigma P3813; Sigma–Aldrich, Missouri, USA) was used to prepare antibody solutions. MilliQ water (Millipore, 18 M) was used for dilution and sample preparation. Sterile and disposable centrifuge tubes (50 mL and 15 mL), pipettes (10 mL and 5 mL) and petri dishes were used for bacteria and virus culture, and sample preparation. Sterilized porcelain spot test plates (Thomas Scientific, 118 × 91 mm 12well, 20.68 mm) were used to perform different steps of the paper based sandwich ELISA test. Each well of the porcelain spot test plates was filled up with 0.5 mL of required solvent, reagent or buffer.

pendent disk shape to avoid cross contamination from tweezers. Paper samples were immersed into primary antibody solution for one hour in the spot test plate to allow antibodies to physisorb on the paper surface. Paper samples impregnated with primary antibodies were then immersed into bovine serum albumin (BSA) for two minutes to block the free paper surface. BSA is used as a blocking agent to block surfaces from antibody adsorption [42], and our study also showed that using BSA could block antibody adsorption on paper by more than 80% (Fig. S2; supporting information). The paper samples were then thoroughly rinsed for one minute using PBS buffer to remove extra antibody and BSA. Serial dilutions (10−1 through 10−10 ) of T7 culture were prepared. Primary antibody and BSA treated paper samples were then immersed into T7 phage solutions of different concentration (100 , 103 , 105 , 107 and 109 pfu/mL, respectively) for 10 min to allow T7 phage interaction with primary antibody and to form primary antibody-T7 phage complexes. The paper samples were then thoroughly washed using PBS buffer to remove unattached phages. The paper samples were then deposited into a HRP conjugated secondary antibody solution for ten minutes. This secondary antibody attached to the antibody-T7 phage complex and formed an antibody-T7 phageantibody sandwich complex. Non-attached secondary antibodies were washed out using PBS buffer. At the end, the paper samples were soaked into a DAB solution for a few seconds and left in a dark chamber for 2 h. DAB reacted with the HRP conjugated to the sandwich complex and produced a brown colour complex as an indication of the presence of active HRP enzyme. Prior to scanning the colour product, the paper samples were washed and left to dry. The samples were then analyzed using standard image processing software. Each reported measurement is the average of 5 full replicates.

3.2. Methods

3.2.3. Image analysis The colour product formed from the HRP-DAB reaction on paper surface was measured at 1200 dpi using a standard scanner (Canon MX410 series Printer and Scanner). The scanned images were analyzed using ImageJ software (ImageJ 1.47t). ImageJ calculates the grey values of RGB images. RGB pixels are converted to grey values using the built in formula (grey = (red + green + blue)/3). For any selected area, the ImageJ programme calculates the weighted average grey value within the selection, which can be related to the enzymatic activity on the paper surface. Thus the average grey value is the sum of the grey values of all the pixels in the selection divided by the number of pixels. The RGB colour scheme uses a numbering scale ranging from 0 to 255 where the ‘black’ is numbered 0 and the ‘white’ is numbered 255, which means the lower the number, the darker the colour. On the other hand, in the CMY (cyan-magenta-yellow) colour scheme the ‘black’ is numbered 255 and the ‘white’ is numbered 0, which means the higher the number the darker the colour. In an enzyme-substrate reaction, high concentration of enzymes yields darker colour, i.e. the darker the colour, the higher the enzyme concentration. To correlate the intensity of colour formation from enzymesubstrate reaction with the colour scheme numbering, the RGB grey values were converted to the CMY grey values: CMY grey values = 255 – RGB grey value. Therefore, the grey value results presented in this article are CMY grey values, i.e., the darker the colour, the higher the enzyme concentration, and the higher the grey values. To develop the experimental curves and plots, the colour signals produced from the enzyme-substrate reaction on paper surface were scanned and analyzed using ImageJ software; the RGB values of the scanned images were converted to CMY values. The CMY values of the blank samples were considered as the baselines (i.e.

3.2.1. T7 phage culture and titre count Escherichia coli BL21 culture at log phase (OD520 = 0.5–1.0) was used as the bacterial host to propagate T7 bacteriophages. The log phase, also known as the exponential phase, is when the bacteria has adapted to the nutrients and environment and start to grow/duplicate in the new setting. LB broth solution (25 g/mL) was used as the growth medium for both bacterial host and T7 bacteriophage cultures. The size distribution of filtered T7 culture was analyzed using dynamic light scattering (DLS), and the results are given in the supporting information (Fig. S1). The concentration of T7 phages was calculated using the bacteriophage plaque assay. For this assay, the agar plates were prepared using 15 mL of hard agar solution (15 g agar in 1 L LB broth solution). Serial dilutions (10−1 through 10−10 ) of T7 culture were prepared, and for each dilution, 0.1 mL of T7 phage sample was mixed with 0.3 mL of host bacteria culture in 3 mL soft agar solution (7 g agar in 1 L LB broth solution), poured onto a hard agar plate, allowed to harden, and incubated overnight at 37 ◦ C to form plaques (for T7 phage culture and plaque assay protocols, see supporting information). Sterile conditions were maintained in every step of culture preparation and plaque assay. The titre count of the cultured T7 was 2 × 109 pfu/mL. 3.2.2. Paper based Sandwich ELISA Researchers have reported different techniques (physisorption, polymer coating, covalent binding, inkjet printing, etc.) [38–41] to deposit biomolecules on paper surface. In this study, primary antibody was physisorbed on paper surface. Fig. 2 shows the experimental steps of developing a paper based sandwich ELISA technique. Filter paper samples (∼1 cm dia) were cut into

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Fig. 2. Experimental steps of paper based sandwich ELISA technique.

origin: 0). The CMY values corresponding to other samples were modified with respect to the corresponding origins. 4. Results

results show strong brown colour formation in the presence of T7 phage solution (Fig. 4). The colour signal is faint for the blank solution. The strong colour for the T7 phage solution confirms the validity of virus detection using this paper based sandwich ELISA technique.

4.1. Enzymatic reaction on paper 4.3. Detection of T7 Phages of different concentrations The time required to complete colour formation reaction for the modified sandwich ELISA technique was measured. A calibration curve was also built to qualify the extent of product colour intensity of HRP-DAB reaction with respect to the HRP concentration. Fig. 3a shows the time required to complete HRP-DAB colour formation reaction for the paper based sandwich ELISA technique. The antibody paper samples were immersed within HRP conjugated secondary antibody for 1 h. The paper samples were then soaked into DAB solution and left in a dark chamber. The HRP-DAB reaction was allowed to run for different time intervals: 0–120 min. To stop the reaction at any time interval, the paper samples were washed in PBS. The colour formed from the HRP-DAB reaction was found to reach the saturation point within 30 min (Fig. 3a). Fig. 3b shows the calibration curve produced for different HRP concentrations. The paper samples were treated with HRP conjugated secondary antibody solutions: 0, 5, 10, 15, 20 ␮g/mL. The colour intensity was found to increase with enzyme concentration on paper. For Fig. 3a and b, error bars indicate standard deviation (n = 5), and no trend lines were used to fit the curves. 4.2. Qualitative detection of T7 phages using antibody active papers The concept of detecting T7 phages using paper based sandwich ELISA was qualitatively verified. Paper samples impregnated with primary antibodies were separately treated with the high concentration of T7 phage solution (2 × 109 pfu/mL) and a blank solution (LB broth solution with no phage), followed by treating with HRP conjugated secondary antibodies and DAB substrate. Experimental

The paper based sandwich ELISA technique was used to quantify a T7 phage concentration in solution. Paper samples impregnated with primary antibodies were separately treated with T7 phage solutions of different concentrations: 0 (blank), 2 × 103 , 2 × 105 , 2 × 107 and 2 × 109 pfu/mL, followed by treating with HRP treated secondary antibodies and DAB substrate. The paper based sandwich ELISA produced colour signals for T7 phages of different concentration. The colour signals formed for different concentration of T7 phages were scanned and analyzed using ImageJ software; The corresponding CMY values of the colour signals were plotted against the T7 phage concentration in semi-logarithmic scale (Fig. 5a). The experimental results produce a calibration curve showing that the colour signal intensity from paper based sandwich ELISA test increases with T7 phage concentration in solution. The experimental results also produce a colour gradient indicating T7 phage concentration (Fig. 5b): the higher the concentration, the darker the colour product formation. The schematic profile represents a possible smoother colour gradient based on the T7 concentration (Fig. 5b). To fit the concentration readings into the logarithmic scale, the concentration of the blank solution was considered 100 instead of 0 (Fig. 5). Using the calibration curve or the colour gradient, the T7 concentration as small as 100 pfu/mL can be detected. The compatibility of paper based sandwich ELISA to conventional titre count was tested using two T7 phage solutions of unknown concentration. Paper based sandwich ELISA tests were conducted for the T7 phage solutions of unknown concentrations; the colorimetric signals produced during the paper ELISA test were analyzed using the calibration curve (Fig. 5). From the logarithmic scale of

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Fig. 3. Enzymatic reaction on paper. (a) Time required to complete colour formation for HRP conjugated secondary antibody treated paper, (b) calibration curve of grey values of the colour product formed from HRP-DAB reaction on paper surface; n = 5, error bars indicate standard deviation.

Fig. 5. Detection of T7 phage concentrations using paper based sandwich ELISA test. (a) The calibration curve shows that the colour signal from paper based sandwich ELISA increases with T7 phage concentrations. n = 5, error bars indicate standard deviation. (b) The colour gradient shows the higher the concentration, the darker is the colour. (c) Detecting unknown concentrations of T7 solutions. The paper based sandwich ELISA results (shown in blue) were compared with the titter count results (shown in black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Fig. 4. Detection of T7 phages using paper based sandwich ELISA test. For the T7 phage sample, the paper based ELISA test gives a strong colour signal. The colour formation is faint for the blank solution.

the calibration curve the concentration of the T7 phage solutions were found 7.53 and 3.81 respectively. From the titre count, the concentrations of the T7 phage solutions in logarithmic scale were found 7.3 and 3.3 respectively (Fig. 5c). 5. Discussion The ELISA technique is mostly used to qualitatively detect the presence of hormones, peptides, antibodies and antigens in blood,

urine and other body fluids. ELISAs are performed using 96-well or 384-well polystyrene plates, which are analyzed using digital plate readers [16,33,34,43]. Researchers have demonstrated that paper based direct ELISA can detect antibody (rabbit IgG), HIV1 antigens, bacteria and viruses [16,18,20,32,44]. Scientists have reported detecting M13 viruses using paper based direct ELISA method, which requires a sophisticated setup including a Bio-Dot filtration apparatus, an ELISA microtitre plate, and a vacuum pump [20]. However, the direct ELISA technique has a lower sensitivity [32]. The limit of detection (LOD) of the modified paper based direct ELISA technique was 106 –107 pfu/mL; to improve the sensitivity (as low as 104 pfu/mL) of the technique, high sample volume

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(100 mL) was required [20]. A paper based diagnosis needs to be sensitive, should be simple in performance and require fewer steps, and should have the capacity of being performed at rural areas with minimum resources and training [1,4,18]. Hence, there is still a need for developing paper based virus detection techniques, which will be highly sensitive (LOD < 105 pfu/mL), will require minimum volume of biofluid, and which can be performed at rural areas with minimum training and equipment. Sandwich ELISA is an indirect ELISA technique which has a few advantages over the direct ELISA technique, such as maximum reactivity of primary antibody and increased sensitivity [33]. Sandwich ELISA technique is two to five times more sensitive than conventional direct ELISA or indirect ELISA techniques [33,45]. The conventional sandwich ELISA technique involves plate preparation using primary antibody. Researchers have demonstrated that antibodies can be physisorbed or ink jet printed on paper, and can be used for further applications [1–3,46]. In this study, the paper was used as an alternative surface to perform the sandwich ELISA test to detect viruses. Because of the infectious characteristics, virus detection using paper diagnostics is more challenging compared to the detection of bacteria, enzymes, DNA or antigens. For the safe execution of the experimental study, the (non-pathogenic) T7 bacteriophage was used in this study. In this technique, antiT7 tag monoclonal IgM antibodies were used as the primary and secondary antibodies. IgM antibodies are star shape pentamers carrying 10 antigen binding sites. Both antibodies are tagged with the protein shell of T7 phage (T7 capsid protein), which ensures the T7 phage interactions with both primary and secondary antibodies and consequently forming sandwich layers. Unlike the conventional techniques, the paper based sandwich ELISA technique can be used for the qualitative and quantitative detection of T7 viruses in solution. For many laboratory diagnosis techniques, the pathogen detection limit varies between 2 × 105 to 2 × 107 units per mL, where value below 2 × 105 units per mL is considered as a ‘negative’ reading [26,47,48]. To detect pathogens as low as 200 units per mL, more sophisticated ‘quantitative’ test is required [47,48]. Our experimental results show that the paper based sandwich ELISA technique forms a colour gradient to quantify the T7 phage concentration in solution (Fig. 5). In this technique, the darker the colour product formed on paper surface, the higher the T7 phage concentration in the solution, and vice versa. The paper based sandwich ELISA technique detected T7 phage concentrations as low as 100 pfu/mL and as high as 109 pfu/mL. Thus the sensitivity of the paper based sandwich ELISA is comparable to the sophisticated laboratory diagnostic techniques. High background and variation of signal are two possible limiting factors of the paper based ELISA technique. In this study, for the blank solution (no T7 phage) the paper based sandwich ELISA did not give a fully white background; rather it gave a light brown colour signal (Fig. 4). Possible reasons for the background are: long incubation time, insufficient blocking, insufficient washing, nonspecific adsorption of HRP conjugated secondary antibodies, and/or colour produced from the substrate. However, the signal intensities for different phage concentrations were stronger than that for the blank solution, and consequently, the colour signalled for T7 phage concentrations were easily visible, detectable and measurable (Fig. 5). For image processing and developing calibration curve, the grey value of the background was measured and subtracted from the grey value of colour signals for different T7 phage concentrations. The signal intensity of the paper based ELISA test depends on the enzyme-substrate concentration, reactivity and purity. The enzyme activity of the HRP conjugated secondary antibody solution was 0.5 U/mL, which hydrolyzes 0.5 ␮mol of substrate per minute. The 3,3 -diaminobenzidine (DAB) tablets used to prepare peroxidase substrate react with HRP on the paper surface and forms a brown

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colour. A second substrate system: liquid DAB (Sigma D3939), was used to check the reproducibility of the enzyme-substrate reaction. The second substrate system formed product colours on paper of different intensities for the same enzyme activity (results not shown), which implies that the signal intensity can vary with substrate activity or concentration. However, for a different set of enzyme and/or substrate system the paper based technique is expected to follow a similar colour trend. This study reports the early generation paper based sandwich ELISA technique for the qualitative and quantitative detection of viruses. This technique also can be integrated as a part of telemedicine [6] with the help of a camera and image processing software, such as ENREF36 [49]. This technique opens a new possibility to researchers and can lead to the development of different sophisticated pathological tests, presently restricted to wellequipped pathological laboratory, using antibody active papers and paper based ELISA techniques. 6. Conclusion Different waterborne and airborne viral diseases cause the death of millions of people every year. Right diagnosis and early detection of those diseases and corresponding viruses can prevent epidemic health threats in many parts of the world. A paper based sandwich ELISA technique can be used to develop paper based diagnostic devices to detect viruses. This paper based technique is sensitive, disposable and biodegradable, which is cheaper and requires less time than the conventional ELISA technique. This study demonstrates that the paper based sandwich ELISA technique can be applied for the qualitative and quantitative detection of a wide range of T7 phage concentrations. With proper calibration and reagents, paper based sandwich ELISA can produce compatible results to traditional titre count. Paper based sandwich ELISA is faster and economic compared to the traditional techniques. Following the biosafety regulations, and with proper training, this technique can be used in remote places to diagnose antigens, virus and other pathogens from water and body fluids. Paper based sandwich ELISA technique combined with the camera of a smart phone or a tablet computer and especially developed smart phone application can be integrated as a part of telemedicine. Acknowledgement This research was supported by SENTINEL Bioactive Paper Network funded by the NSERC-CRSNG (for details, see: www. bioactivepaper.ca), and NSERC Discovery Grants. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.05. 028 References [1] M.S. Khan, et al., Paper diagnostic for instantaneous blood typing, Anal. Chem. 82 (10) (2010) 4158–4164. [2] M. Al-Tamimi, et al., Validation of paper-based assay for rapid blood typing, Anal. Chem. 84 (3) (2012) 1661–1668. [3] M. Li, et al., Paper-based blood typing device that reports patient’s blood type “in Writing”, Angew. Chem. Int. Ed. 51 (22) (2012) 5497–5501. [4] R. Pelton, Bioactive paper provides a low-cost platform for diagnostics, Trends Anal. Chem. 28 (8) (2009) 925–942. [5] S. Aikio, et al., Bioactive Paper and Fibre Products: Patent and Literature Survey, VTT Working Papers 51, 2006, pp. 1–84. [6] A.W. Martinez, et al., Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis, Anal. Chem. 80 (10) (2008) 3699–3707.

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M.S. Khan et al. / Colloids and Surfaces B: Biointerfaces 132 (2015) 264–270

[7] S.M.Z. Hossain, et al., Development of a bioactive paper sensor for detection of neurotoxins using piezoelectric inkjet printing of sol-gel-derived bioinks, Anal. Chem. 81 (13) (2009) 5474–5483. [8] R. Pelton, in: S.J. I’Anson (Ed.), Bioactive Paper – A Paper Science Perspective, in Advances in Pulp and Paper Research, Oxford 2009, The Pulp & Paper Fundamental Research Society, 2009, pp. 1096–1145. [9] M.S. Khan, G. Garnier, W. Shen, Printing, Specificity and Stability of Bioactive Papers, VDM Publishing House, Saarbrucken, Germany, 2010. [10] J. Tian, et al., Capillary driven low-cost v-groove microfluidic device with high sample transport efficiency, Lab Chip 10 (17) (2010) 2258–2264. [11] S.M.Z. Hossain, et al., Multiplexed paper test strip for quantitative bacterial detection, Anal. Bioanal. Chem. 403 (6) (2012) 1567–1576. [12] M.M. Ali, et al., Detection of DNA using bioactive paper strips, Chem. Commun. (43) (2009) 6640–6642. [13] Y. Akahori, et al., An alcohol gas – sensor using an enzyme immobilized paper, Chem. Sens. 20 (Suppl. B) (2004) 468–469. [14] G. Masao, Preparation of Enzyme-Immobilized Filter Paper for Determination of Freshness of Fish Meat, Jpn Kokai Tokkyo Koho, Japan, 1989. [15] J. Tian, X. Li, W. Shen, Printed two-dimensional micro-zone plates for chemical analysis and ELISA, Lab Chip 11 (2011) 2869–2875. [16] C.-M. Cheng, et al., Paper-based ELISA, Angew. Chem. Int. Ed. 49 (28) (2010) 4771–4774. [17] M. Cavaiuolo, et al., Development and optimization of an ELISA based method to detect listeria monocytogenes and Escherichia coli O157 in fresh vegetables, Anal. Methods 5 (18) (2013) 4622–4627. [18] M.N. Costa, et al., A low cost, safe, disposable, rapid and self-sustainable paperbased platform for diagnostic testing: lab-on-paper, Nanotechnology 25 (9) (2014) 1–12. [19] H. Shafiee, et al., Paper and flexible substrates as materials for biosensing platforms to detect multiple biotargets, Sci. Rep. 5 (2015). [20] P.A. Larsson, et al., Filtration, adsorption and immunodetection of virus using polyelectrolyte multilayer-modified paper, Colloids Surf. B: Biointerfaces 101 (0) (2013) 205–209. [21] C. Parolo, A. Merkoci, Paper-based nanobiosensors for diagnostics, Chem. Soc. Rev. 42 (2) (2013) 450–457. [22] WHO, Hepatitis B, World Health Organization, Geneva, Switzerland, 2002, pp. 76. [23] WHO, Hepatitis A, World Health Organization, Geneva, Switzerland, 2000, pp. 41. [24] UNAIDS, UNAIDS World AIDS Day Report 2012, UNAIDS, Geneva, Switzerland, 2012, pp. 48. [25] WHO, Hepatitis Delta, World Health Organization, Geneva, Switzerland, 2001, pp. 30. [26] W. Preiser, C. Drosten, in: B.S. Kamps, C. Hoffmann (Eds.), SARS Reference – 10/2003, Flying Publisher, 2003, p. 172. [27] UNICEF/WHO, Diarrhoea: Why Children are Still Dying and What Can Be Done, UNICEF/WHO, New York, USA/Geneva, Switzerland, 2009, pp. 41.

[28] WHO, Polio’s Last Stand? WHO: Independent Monitoring Board of the Global Polio Eradication Initiative, Geneva, Switzerland, 2012, pp. 50. [29] ATCC, Product Information Sheet for Escherichia Coli Bacteriaphage T7 ATCC® BAA-1025-B2, Manassas, VA, USA, 2013. [30] abcam, Product Datasheet of anti-T7 tag® antibody (ab50545). Cambridge, UK. [31] Sigma, Product Information of Monoclonal Anti-T7 tag Peroxidase Conjugate (T3699), Missouri, USA, 2003. [32] J.M. Walker (Ed.), The ELISA Guidebook, 2nd ed., Human Press, 2009. [33] KPL, ELISA Technical Guide, Gaithersburg, MD USA, 2013. [34] M. Harboe, et al., Antibody response in rabbits to immunization with mycobacterium leprae Infect. Immun. 18 (3) (1977) 792–805. [35] Peroxidase from Horseradish (HRP), in Product Information. Sigma–Aldrich. (web: www.sigmaaldrich.com). [36] N.C. Veitch, Horseradish peroxidase: a modern view of a classic enzyme, Phytochemistry 65 (3) (2004) 249–259. [37] Sigma, Product Information of SIGMAFASTTM 3,3 -Diaminobenzidine Tablets (D4168), Missouri, USA, 2007. [38] M.S. Khan, et al., Effect of polymers on the retention and aging of enzymatic bioactive papers, Colloids Surf. B: Biointerfaces 79 (1) (2010) 88–96. [39] L. Manfredi, R.J. Hill, T.G.M.v.d. Ven, Bridging flocculation of PEI-functionalized latex particles using nanocrystalline cellulose, J. Colloid Interface Sci. 360 (1) (2011) 117–123. [40] C. Venkataprasad, Adsorption of Herbicides and Bacteria Using Pulp Fibers, in Mechanical and Industrial Engineering, Master of Applied Science Thesis, Concordia University, Montreal, Canada, 2009, pp. 1–58. [41] M.S. Khan, et al., Thermal stability of bioactive enzymatic papers, Colloids Surf. B: Biointerfaces 75 (1) (2010) 239–246. [42] Z. Xia, H.L. Goldsmith, T.G.M.v.d. Ven, Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds, Biophys. J. 66 (1994) 1222–1230. [43] Thermo Scientific Pierce Assay Development Technical Handbook, Thermo Fisher Scientific Inc., 2011, pp. 76. [44] K. Zhu, et al., Recent developments in antibody-based assays for the detection of bacterial toxins, Toxins 6 (4) (2014) 1325–1348. [45] S.R. Mikkelsen, E. Corton, Bioanalytical Chemistry, John Wiley & Sons, Inc., Publication, New Jersey, 2004, pp. 361. [46] P. Jarujamrus, et al., Mechanisms of red blood cells agglutination in antibodytreated paper, Analyst 137 (9) (2012) 2205–2210. [47] A. Ernst, Hepatitis Central: Viral Load, 2013, Available from: http://www. hepatitiscentral.com/hepatitis-c/what-is-viral-load.html (13.04.15). [48] G. López-Campos, et al., Detection, Identification, and Analysis of Foodborne Pathogens, in Microarray Detection and Characterization of Bacterial Foodborne Pathogens, Springer, US, 2012, pp. 13–32. [49] M.S. Khan, G. Garnier, Direct measurement of alkaline phosphatase kinetics on bioactive paper, Chem. Eng. Sci. 87 (2013) 91–99.

Qualitative and quantitative detection of T7 bacteriophages using paper based sandwich ELISA.

Viruses cause many infectious diseases and consequently epidemic health threats. Paper based diagnostics and filters can offer attractive options for ...
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