Author’s Accepted Manuscript Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk Ling Yu, ZhuanZhuan Shi, Can Fang, YuanYuan Zhang, YingShuai Liu, ChangMing Li www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(15)00136-0 http://dx.doi.org/10.1016/j.bios.2015.02.035 BIOS7484
To appear in: Biosensors and Bioelectronic Received date: 5 December 2014 Revised date: 10 February 2015 Accepted date: 23 February 2015 Cite this article as: Ling Yu, ZhuanZhuan Shi, Can Fang, YuanYuan Zhang, YingShuai Liu and ChangMing Li, Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.02.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Disposable Lateral Flow-through Strip for Smartphonecamera to Quantitatively Detect Alkaline Phosphatase Activity in Milk Ling Yu, a,b,c ZhuanZhuan Shi, a,b,c Can Fang,d YuanYuanZhang,a,b c YingShuai Liua,b,c and ChangMing Li *a,b,c a
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy,
Southwest University, Chongqing 400715, China b
Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies,
Chongqing 400715, China c
Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease,
Southwest University, Chongqing, 400715, China d
School of Computer and Information Science, Southwest University, Chongqing,
400715, China *Corresponding authors: Chang Ming Li (C.M.Li)Tel: +86-23-68254842; E-mail: [email protected]; [email protected]
Abstract A disposable lateral flow-through strip was developed for smartphone to fast onestep quantitatively detect alkaline phosphatase (ALP) activity in raw milk. The strip comprises two functional components, a conjugation pad loaded with phosphotyrosine-
1
coated gold nanoparticles (AuNPs@Cys-Try-p) and a testing line coated with antiphosphotryosine antibody (anti-Tyr-p mAb). The dephosphorylation activity of ALP at the testing zone can be quantitatively assayed by monitoring the accumulated AuNPsinduced colour changes by smartphone camera, thus providing a highly convenient portable detection method. A trace amount of ALP as low as 0.1 U L-1 with a linear dynamic range of 0.1-150 U L-1 (R2=0.999) in pasteurized milk and raw milk can be onestep detected by the developed flow-through strip within 10 min, demonstrating the potential of smartphone-based portable sensing device for pathogen detection. This biohazards free lateral flow-through testing strip
can be also used to fabricate rapid,
sensitive and inexpensive enzyme or immunosensors for broad portable clinic diagnosis and food contamination analysis, particularly in point-of-care and daily food quality inspection.
Key words: lateral flow-through strip, alkaline phosphatase detection, gold nanoparticle, milk, point-of-care detection
Introduction Milk and its derivatives can harbor a variety of microorganisms, which are important sources of foodborne diseases(Klinger and Rosenthal, 1997; Oliver et al., 2005; Oliver et al., 2009). Pasteurization is a standard method to eliminate pathogens. Inadequate or faulty pasteurization will not kill all foodborne pathogens(Angelino et al., 1999; Oliver et al., 2005; Rankin et al., 2010). Alkaline phosphatase (ALP), one of the enzymes existing in raw milk, is a hydrolase enzyme responsible for removing phosphate
2
groups from many types of biomolecules, including nucleotides, proteins, and alkaloids(Gettins et al., 1985). It is slightly less labile to heat (71.6°C for 15 second) than most pathogenic bacteria, and loss of ALP activity is used to confirm proper pasteurization of skimmed or whole milk(Sharma et al., 2003). The level of ALP in milk is variable depending on the source of raw milk, and the permitable value by the U.S. and European countries is 0.35 units/L in pasteurized drinks. Thus, ALP level or activity can be employed to measure degree of pasteurization of drinks, especially milk and its derivatives(Sharma et al., 2003; Oliver et al., 2005; Klotz et al., 2008; Rankin et al., 2010; Albillos et al., 2011). Considering the importance of ALP in food safety and clinical diagnosis, many methods such as spectrophotometry(Bianchi et al., 1994), electrochemistry(Ito et al., 2000; Murata et al., 2009; Santiago et al., 2010; Miao et al., 2011; Jiang and Wang 2012; Zhang et al., 2013), chemiluminescence(Blum et al., 2001; Salter and Fitchen 2006; Albillos et al., 2011) and colorimetric assays (Sharma et al., 2003; Choi et al., 2007; Li et al., 2013) have been developed to analyze ALP. Although spectrometric method is sensitive and accurate it requires expensive instruments, professional operators, timeconsuming procedures, complicated sample processes and large amount sample, greatly limiting its applications for in-field food inspection(Bianchi et al., 1994; Thompson et al., 1991). Electrochemical and chemiluminescence methods have been explored to achieve sensitive detection of ALP(Ito et al., 2000; Murata et al., 2009; Santiago et al., 2010; Miao et al., 2011; Jiang and Wang 2012; Zhang et al., 2013). However, the former needs complicated instruments for electrochemical detection while the latter relies on desktop equipment.
Even though some of these assays have shown high sensitivity, their
3
applications for in-field testing, especially for rural countries that do not have enough laboratory resources, are very difficult for distant services. Thus, a simple, portable, disposable and quantitative ALP detection tool is highly desired to fulfill daily food safety supervision, especially for under-developed regions. Recently, metal nanoparticles such as gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) have been used as signal reporters in colorimetric assays for ALP detection(Choi et al., 2007; Li et al., 2013). These methods are based on ALP induced distinguishable color changes for qualitative or semi-quantitative detections. UV-vis spectrometer has been used for quantitative analysis, but hindering point-of-care application. A reflectoquant® ALP test kit has been developed by Merck to evaluate pasteurization of milk. However, an eight-step sample handling process is needed and the detection has to be performed under a specific temperature (37°C)( Merck KGa-D64271, 2002). Obviously such a test kit is only suitable for ALP analysis in special labs. In vitro lateral flow strips based on the principle of immunochromatography have been widely employed to detect different chemical and biochemical molecules in various applications(Glynou et al., 2003; Mao et al., 2009; Xu et al., 2009; Li et al., 2010; Drygin et al., 2012; Liu et al., 2012). This method allows a one-step, rapid and inexpensive analysis by end users. Sharma et al. have developed a dry-reagent strip to examine ALP activity, which is based on ALP reaction with p-nitrophenyl phosphate in the presence of water to liberate p-nitrophenol and inorganic phosphate. Then p-nitrophenol reacts with a specific chromogen to change the color of the strip from light blue to green, which can be visualized by naked eyes(Sharma et al., 2003). This strip is a good candidate for remote
4
or rural areas where expensive instruments are not available. Unfortunately, pnitrophenyl phosphate may cause eye, skin and respiratory tract irritation(Fisher Scientific 2012), while p-nitrophenol is extremely hazardous in case of ingestion and very hazardous in case of skin and eye contact as well as inhalation(Gao et al., 1997). In addition, the capability for quantitative analysis by this dry-reagent strip has not been disclosed. Herein, we report a lateral flow-through strip to detect the activity of ALP by using phosphotyrosine-capped AuNPs (AuNPs@Tyr-p) as a signal reporter. The accumulation of AuNPs at the testing zone results in a purple-brown color that can be clearly observed by naked eyes. The images are captured by a smartphone camera and are further analyzed by a home-programmed MATLAB® code to sensitively and quantitatively monitor ALP in raw cow milk. The results clearly show that the developed bio-hazards free, lateral flow-through strip is capable of portable in-field detection of ALP. 2. Materials and methods 2.1 Materials HAuCl4, sodium citrate, anti-phosphotyrosine antibody, O-Phospho-L-tyrosine, alkaline
phosphatase,
L-cysteine,
glutaraldehyde,
NaBH4,
polyethylene
glycol
(MW:20000), bovine serum albumin were purchased from Sigma Aldrich. Dialysis bag (8000-14000D) were received from scientific research special (USA). Glass fiber (SB08), polyester fiber paper (DL 42), nitrocellulose membrane (sartorius CN140), adsorbent pad (SX27) and polyvinyl chloride (PVC) backing card were ordered from Shanghai Kinbio
5
Tech (China). All other chemicals used in this study are analytical grade. The deionized (DI) water used in all experiments was produced by PURELAB flex system, ELGA Corporation. 2.2 Preparation of AuNPs@Cys-Tyr-p conjugates In a typical experiment, 2 mL 1% (m/V) HAuCl4 (diluted in DI water) was mixed with 198 mL DI water in a 500 mL flask. The thoroughly mixed HAuCl 4 solution was boiled in an oil bath. Then 4 mL 1% (m/V) sodium citrate (diluted in DI water) was added into boiled HAuCl4 solution for around 30 min, when color of the solution turned to red and no further color change occurred. A two-step conjugation approach was conducted to prepare Au nanoparticles@Cysteine-phosphoTyrosine (AuNPs@Cys-Tyr-p) conjugates (Scheme1). First, 10 mL L-cysteine (1mg mL-1) was added into 10 mL AuNPs solution under continuous stirring at room temperature (RT) for 2 h. The mixture was dialyzed with DI water at 4°C for 10 h by replacing the DI water every 2 h to obtain cysteine functionalized AuNPs (AuNPs@Cys). 5 mL O-Phospho-L-tyrosine (500 μM) was mixed with 5 mL AuNPs@Cys solution. Then 100 μL glutaraldehyde solution (0.25% in DI water, v/v) was slowly dropped into O-Phospho-L-tyrosine and AuNPs@Cys mixture, followed by 1 h stirring at RT. 50 μL 0.1% (w/w) NaBH4 was added into the above mixture to reduce Schiff’s base formed between aldehyde and amine. Finally, the product was dialyzed with DI water for 10 h and then concentrated with polyethylene glycol (WM 20000) to 5 mL. The harvested phosphor-tyrosine conjugated AuNPs (AuNPs@Cys-Tyr-p) was characterized and used for lateral flow-through strip construction.
6
Scheme 1.
2.3 Characterization of AuNPs@Cys-Tyr-p conjugates Morphology of AuNPs was characterized using a field emission scan electron microscope (FESEM, JSM-7800F). For FESEM analysis, the prepared AuNPs solution was drop-cast on a silicon wafer and allowed drying in open atmosphere. UV-vis spectra of
pristine
and
modified
AuNPs
were
recorded
using
Shimadzu
UV-vis
spectrophotometer (UV-2550, Japan). Zeta potential measurements were performed with Malvern Zetasizer Nano ZS 90 (Malvern, UK) equipped with a standard 633 nm laser. Binding energies were measured by X-ray photoelectron spectroscopy (XPS) (XPS, Thermo, USA) with C1s binding energy at 285.05eV as a reference. The sample for XPS characterization was prepared by placing 100 μL of the prepared solutions on a clean glass slide. 2.4 Assembling of ALP detection lateral flow-through strip The test strip consists of a sample-loaded pad, AuNPs conjugation pad, nitrocellulose membrane, adsorbent pad and backing card (Scheme 2). The sample loading pad (20 mm×4 mm) was made from a glass fiber paper and the conjugation pad (8 mm×4 mm) was made of a polyester fiber paper. The test zone was prepared by dispensing of 1.5 μL of 2 mg mL-1 anti-phosphotyrosine monoclonal antibody (anti-Tyr-p mAb) onto the nitrocellulose membrane (25 mm×4 mm). After drying at room temperature (RT) for 1 h, the membrane was blocked with 2% BSA for 30 min and then
7
dried at RT. Then, all of the parts were assembled on a plastic adhesive backing card, and each part overlapped 2 mm to ensure the solution migrating through the entire strip during assays. Finally, the strip was assembled into a strip cassette for further use. 2.5 ALP detection on lateral flow-through strip The sample solution containing a desired concentration of ALP was added onto the sample pad, in which the heat-denatured ALP was used here as a negative control. After about 10 min, the test strip was imaged by a smartphone integrated digital camera (vivo S7i, 5×106 pixels). All experiments were repeated at least three times independently. 2.6 Colorimetric changed analyzed by NIH ImagJ The grey value of the testing pad was quantified by NIH free software ImageJ. For each strip, to eliminate the influence of uneven illumination, the AuNPs accumulation induced colormetric change can be quantified by taking a calculation defined below: G.I.= [(A1-A0)/A0]×100%
(1)
where G.I. is the normalized grey intensity of the testing zone, A1 is the grey value of the mAbs immobilization zone, and A0 is the grey value of neighborhood unreacted/non Ab immobilized area of the strip. Through this calculation, the more AuNPs captured by mAbs at the testing zone, the larger G.I. is obtained. 2.7 Detection strategy Scheme 2 illustrates the configuration and principle of the AuNPs based lateral flow strip. During the assay, an aqueous sample containing active ALP was dropped on
8
the sample loading pad as shown in Scheme 2A (step1). Subsequently, the analyte migrates along the strip via capillary action and then reacts with AuNPs@Cys-Tyr-p in the conjugation zone. Active ALP can convert AuNPs@Cys-Tyr-p to AuNPs@Cys-Tyr by removing phosphate groups (step 2). AuNPs@Cys-Tyr cannot be captured by antiTyr-p mAb, thus no color changes can be observed at test line (step 3). A sample without ALP or with denatured ALP cannot remove the phosphate group of AuNPs@Cys-Tyr-p, which are seized by anti-Tyr-p mAb, resulting in a visual band because of the accumulation of AuNPs at test line (Scheme 2B). Therefore the ALP activity is reflected by the color changes at the testing line. In this case, the more active ALP in the sample, the less AuNPs conjugates would be captured at the test line. Sample containing active ALP will give a “white or light” line, while negative sample will give a “pink/red or dark” line. According to the principle described above, grey intensity on the test line would be reversely proportional to active ALP in samples. Scheme 2.
2.8 Statistical analysis Results were provided as means ± standard errors (SE). The data were analyzed by Student’s t-test using Origin Statistic software (Origin Lab Corporation, USA). A p-value < 0.05 was considered significant. 3. Results and discussion 3.1 Amino acid conjugated AuNPs
9
Preparation of AuNPs@Cys-Tyr-p with good size-distribution and dispersion is the key factor for successfully fabrication of nanoparticle based flow-through strip. A twostep conjugation strategy as illustrated in Scheme 1 was applied to synthesize phosphotyrosine functionalized AuNPs. Table 1 summaries the reaction condition and the quality of obtained amino acid-functionalized AuNPs.
The first step is to prepare
cysteine-functionalized AuNPs (cysteine@AuNPs) by simply mix AuNPs and cysteine solution. It was found that a lower concentration of cysteine will not lead to aggregation after the reaction mixture was dialyzed against DI water (pH=6.5). This phenomena is well in-line with the reported literature (Majzik et al., 2009).
During the second
conjugation step, O-phospho-L-tyrosine is linked to cysteine@AuNPs by cross-linker glutaraldehyde. The conjugation condition was titrated to avoid high concentration of amino acid and glutaraldehyde induced particle aggregation (Migneault et al., 2004). The results show that O-phospho-L-tyrosine (500 μM, 5mL) mixed with 5 mL cysteine@AuNPs and glutaraldehyde (0.25%, 100 μL) for 1 h will no lead to AuNPs aggregation. Table 1 With the optimized condition, AuNPs@Cys-Tyr-p was synthesized for property characterization and lateral flow-through strip fabrication. Field emission scanning electronic microscopy (FESEM) result (Fig.1A) shows that the synthesized AuNPs are uniform. The UV-vis absorption spectra of the AuNPs, AuNPs@Cys, and AuNPs@CysTyr-p (Fig. 1B) reveal that the adsorption peak is narrow and close to the AuNPs’ surface plasmon resonance range (~520 nm). The adsorption peak of AuNPs is at 519 nm
10
(Fig. 1A, a). Conjugation with cysteine results in a red shift of the adsorption peak to 525 nm (b). While AuNPs@Cys-Tyr-p gives an adsorption peak centered at 530 nm (c). Furthermore, pristine and modified AuNPs were examined by zeta potential measurements. The zeta-potentials of AuNPs, AuNPs@Cys, and AuNPs@Cys-Tyr-p measured in DI water (Fig. 1C) show that AuNPs@Cys-Tyr-p gives the largest negative value (-34.67 ± 1.35 mV, n=3), which should be resulted from the conjugation of negatively charged phosphate group on the tyrosine. The large potential indicates that AuNPs@Cys-Tyr-p has good dispersibility in an aqueous solution. The particle size analysis indicates that diameter of the bare AuNPs is around 16.16 ± 0.26 nm. With the conjugation of cysteine and phosphotyrosine, sizes of the nanoparticles increase to 17.19 ± 0.84 nm and 21.70 ± 0.93 nm, respectively (Fig. 1D).
Figure 1
XPS analysis in Fig. 2A shows that the typical Au 4f5/2 and Au 4f7/2 doublets with binding energies of 87.3 and 83.6eV are clearly observed, confirming the metallic state of the prepared nanoparticles. The N 1s core-level spectrum with an expected major peak at 400.1eV indicates that the amino acid is successfully attached to AuNPs (Fig. 2B). The P 2p region displays a characteristic peak at 133.2eV, which can be assigned to phosphate group of the O-Phospho-L-tyrosine (Fig. 2C). The C1s core-level spectra can be fitted into four peak components with binding energies at 285.4, 284.7, 286.9 and 288.2eV, attributing to the C-C, C=C, C-O C-S, C-N and
11
, respectively (Fig. 2D).
Figure 2
The combined results from UV-vis absorbance spectra, zeta potentials and XPS characterization leads to a clear conclusion that AuNPs@Cys-Tyr-p was successfully prepared through a two-step conjugation approach, in which cysteine works as a heterobifunctional cross-linker attaches on AuNPs through the interaction of Au and –SH group, while providing–NH2 group to covalently link of phosphotyrosine. The negatively charged phosphate group on tyrosine side chain not only prohibits aggregation of AuNPs, but serves as the target of ALP. It is further proves that during the functionalization process, dialysis instead of centrifugation is successfully used to remove free amino acid for avoiding
AuNPs aggregation. Different batches of AuNPs@Cys-Tyr-p were
quantified by measuring the absorbance at 530 nm. In all remaining experiments, AuNPs@Cys-Tyr-p solution with an absorbance of ~0.27 was used to prepare the ALP lateral flow strip. 3.2 Optimization of lateral flow-through strip The amount of AuNPs@Cys-Tyr-p loaded on conjugation pad and the concentration of antibody on testing zone directly affect colorimetric response of the strip since the signal mainly depends on the amount of AuNPs@Cys-Tyr-p captured by the immobilized antibody on the test line. Different amount of AuNPs@Cys-Tyr-p conjugates and anti-phosphore-tyrosine monoclonal antibody (anti-Tyr-p mAbs) were applied in a chessboard assay (Fig.3A) to find optimal condition for a high signal-tonoise ratio. The difference of the colors in the testing zone can be distinguished by naked 12
eyes. In addition, the grey intensity of the testing zone was quantified by NIH ImageJ as described in Section 2.6. As shown in Fig. 3B, the chessboard result indicates the highest G.I. of 29.4 was resulted from the combination of dispensing 40 μL AuNPs@Cys-Tyr-p conjugates and 2 mg mL-1 anti-phosphotyrosine antibody. In following experiment, the optimal AuNPs@Cys-Tyr-p conjugates and antibody concentrations were used to fabricate ALP lateral flow-through strip. Figure 3
3.3 Smartphone based in-field detection strategy The enthusiasm of the developed AuNPs@Cys-Tyr-p based strip will be enhanced if the testing images can be analyzed by portable devices such as a smartphone. Therefore, a MATLAB® (Mathworks cop, USA) code was developed to quantify optical intensity of the testing zone on the strip. The smartphone acquired images were processed by the following procedures: a. each color image was separated into Red, Green and Blue channel, and all channels were then stacked into one grey scale image by taking a weighted sum. The stacked image was considered as a matrix of brightness values; b. For each strip, the program automatically selected a number of reference spots from unreacted area (non antibody immobilization zone) close to the testing zone (antibody immobilization zone). Reference brightness value (RefB) was calculated by taking the average of all reference spots in each strip; c. For each pixel in testing zone, its brightness signal was normalized by a calculation defined below: Brightness (Br)= (RefB-TestB)/RefB
13
(2)
where TestB is the brightness value of this pixel. The effect of uneven illumination can be minimized by this normalization operation; d .Each row of the normalized matrix was considered as a curve of brightness values. These curves were aligned, stacked into one curve and then smoothed by adopting the moving-polynomial smoother to reduce the noise. Finally, the peaks of each stacked curve are automatically recorded by the program. The typical photographs of control (denatured 100 U L-1 ALP) and 100 U L-1 ALP captured by mobile phone camera were shown in Fig. 4A, clearly indicating that existence of ALP significantly reduces the pink/purple color at testing zone. The smartphone camera captured picture was quantitatively analyzed by the MATLAB® code as described above. The results show that the purple/dark band from sample with denatured ALP is characterized by a peak value of 100 (a.u.). 100 U L-1 active ALP induces a dramatic decrease of peak value (Fig.4 B). Since the MATLAB® code can be programmed to a mobile phone APP, providing an economic and portable quantitative analysis solution for the developed ALP lateral flow strip. The combination of disposable ALP lateral flow-through strip and popular smartphone highlight the potential of daily food sanitation supervision by end user.
Figure 4
3.4 The optimized condition for one-step detection of ALP by AuNPs@Cys-Tyr-p based lateral flow-through strip To achieve a high analytical performance, the assay conditions of pH and temperature were optimized. To quantify the ALP activity induced colorimetric changes,
14
the assayed strips were imaged by smartphone camera. The brightness (Br) of testing zone of each strip was quantified with the strategy detailed in section 3.3 and the ALP activity induced colorimetric changes (ΔBr % ) was calculated as follow: ΔBr % = [(Brcontrol – BrALP)/Brcontrol] × 100%
(3)
where Brcontrol is the normalized brightness of control sample (denatured ALP spiked milk), BrALP is the normalized brightness of strip for measuring ALP-spiked milk. The colorimetric change (ΔBr%) is plotted as a function of different concentrations of ALP in milk. From pictures in Fig.5A, clear pink/purple bands can be observed from strips tested analyte without ALP (control, 0 U/L). Although it is documented that ALP prefers a alkaline condition, the assay buffer with a pH value of 10 does not results in good performance. While, pH 8 favors the capture and binding activity of antibody and might not inhibit the activity of ALP. Therefore, more AuNPs@Cys-Tyr-p can be seized by the anti-Tyr-p mAb and the largest colorimetric changes can be achieved by 1 U/L ALP. To evaluate the effect of temperature on the assay’s performance, the strips and samples were place at 15, 25, 37 and 45°C prior to the tests (Fig.5B). Unexpectedly, 37°C as a physiological favorable condition does not give the highest ALP induced colorimetric change. It may due to the increased non-specific interaction of the amino acid functionalized AuNPs and the mAb. Encouragingly, in terms of practical application, ambient temperature (25°C) will simplify the testing condition because incubation at 37°C or higher temperature that needs specific instrumentation will be avoided. Milk from most mammals normally contains low concentration of vitamin C and lactose, a
15
sugar composed of glucose and galactose molecules. To investigate if the co-existing components may affect the performance of the AuNPs@Cys-Tyr-p based lateral flowthrough strip, ascorbic acid (5 mg/mL), lactose (10 mg/mL) and glucose (2 mg/mL) were spiked into samples containing 1U/L ALP. The quantitative ΔBr% from different anlytes are very close, indicating those biological molecules will not infer the ALP activity induced colorimetric change (Fig.5C). In summary, to reservoir the better biological activity of both antibody and the enzyme, analytes should be keep in a solution with pH value of 8. While to easy the practical application and to reduce the non-specific binding, the assay can be conducted in ambient temperature. Co-existing of vitamin C and sugar will not affect the performance of the strip, demonstrating the good specificity of the assay. With the optimized assay condition, the strip was applied for testing ALP in milk. Figure 5
3.5 Performance of AuNPs@Cys-Tyr-p based lateral flow-through strip in detecting ALP in milk To explore the feasibility of AuNPs@Cys-Tyr-p based ALP strip for milk sanitation inspection, the strip was applied to detect ALP-spiked milk. Because milk contains a large amount of macromolecule, such as protein and lipid, it is important to set up a sample pretreatment condition to achieve a sensitive detection. Centrifugation was used to pre-treat milk for reducing unexpected interference from various components and additives. Fig. 6A shows that centrifugation process (6000 rpm for 5 min at 4°C) sharply
16
increases signal-to-nose ratio. It can be explained that centrifugation can assist separate and remove of lipid component which can affect the migration of AuNPs@Cys-Tyr-p on the lateral flow-through strip. Next, different concentrations of ALP were spiked into 0.1% centrifuged commercial available pasteurized milk and tested by developed strips. Fig.6B shows typical response of the strip within 10 min for ALP at concentrations of 0, 1 and 100 U L-1, respectively. Denatured ALP spiked milk was treated as a negative control. It is observed that the purple/pink color at testing zone become weaker with the increase of ALP concentration (Fig. 6B). To investigate whether the AuNPs@Cys-Tyr-p based strip could provide quantitative detection of ALP in milk, the test zones of strips were imaged by smartphone camera and the brightness (Br) of testing zone of each strip reacted with different amount of ALP was quantified with a home programmed MATLAB® code (formula 2 at section 3.3) and the ALP activity induced colorimetric change (ΔBr%) was calculated as equation (3). The quantified ΔBr % is plotted as a function of different concentrations of ALP in milk. The typical dose-signal response curve was drawn from three independent tests was shown in Fig. 6C. The detection range of the strip covers 0.1150 U L-1 (y= 38.30+21.86x, R2=0.999, n=3). Considering milk sample was diluted 1000-fold, for undiluted milk the detection limit of 0.1 U L-1 is equivalent to 100 U L-1, which is comparable to level of the ALP in raw milk (500-1100 U L-1) (Payne et al., 2009).
Figure 6
17
Activity of ALP in fresh raw cow milk sample collected directly from a farm was tested by the developed ALP lateral flow-through strip. The results show that diluted (0.1%, v/v) raw milk compared to commercial pasteurized milk gives a ΔBr% of 30.27 ± 1.17 (n=3, p
PII: DOI: Reference:
S0956-5663(15)00136-0 http://dx.doi.org/10.1016/j.bios.2015.02.035 BIOS7484
To appear in: Biosensors and Bioelectronic Received date: 5 December 2014 Revised date: 10 February 2015 Accepted date: 23 February 2015 Cite this article as: Ling Yu, ZhuanZhuan Shi, Can Fang, YuanYuan Zhang, YingShuai Liu and ChangMing Li, Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.02.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Disposable Lateral Flow-through Strip for Smartphonecamera to Quantitatively Detect Alkaline Phosphatase Activity in Milk Ling Yu, a,b,c ZhuanZhuan Shi, a,b,c Can Fang,d YuanYuanZhang,a,b c YingShuai Liua,b,c and ChangMing Li *a,b,c a
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy,
Southwest University, Chongqing 400715, China b
Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies,
Chongqing 400715, China c
Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease,
Southwest University, Chongqing, 400715, China d
School of Computer and Information Science, Southwest University, Chongqing,
400715, China *Corresponding authors: Chang Ming Li (C.M.Li)Tel: +86-23-68254842; E-mail: [email protected]; [email protected]
Abstract A disposable lateral flow-through strip was developed for smartphone to fast onestep quantitatively detect alkaline phosphatase (ALP) activity in raw milk. The strip comprises two functional components, a conjugation pad loaded with phosphotyrosine-
1
coated gold nanoparticles (AuNPs@Cys-Try-p) and a testing line coated with antiphosphotryosine antibody (anti-Tyr-p mAb). The dephosphorylation activity of ALP at the testing zone can be quantitatively assayed by monitoring the accumulated AuNPsinduced colour changes by smartphone camera, thus providing a highly convenient portable detection method. A trace amount of ALP as low as 0.1 U L-1 with a linear dynamic range of 0.1-150 U L-1 (R2=0.999) in pasteurized milk and raw milk can be onestep detected by the developed flow-through strip within 10 min, demonstrating the potential of smartphone-based portable sensing device for pathogen detection. This biohazards free lateral flow-through testing strip
can be also used to fabricate rapid,
sensitive and inexpensive enzyme or immunosensors for broad portable clinic diagnosis and food contamination analysis, particularly in point-of-care and daily food quality inspection.
Key words: lateral flow-through strip, alkaline phosphatase detection, gold nanoparticle, milk, point-of-care detection
Introduction Milk and its derivatives can harbor a variety of microorganisms, which are important sources of foodborne diseases(Klinger and Rosenthal, 1997; Oliver et al., 2005; Oliver et al., 2009). Pasteurization is a standard method to eliminate pathogens. Inadequate or faulty pasteurization will not kill all foodborne pathogens(Angelino et al., 1999; Oliver et al., 2005; Rankin et al., 2010). Alkaline phosphatase (ALP), one of the enzymes existing in raw milk, is a hydrolase enzyme responsible for removing phosphate
2
groups from many types of biomolecules, including nucleotides, proteins, and alkaloids(Gettins et al., 1985). It is slightly less labile to heat (71.6°C for 15 second) than most pathogenic bacteria, and loss of ALP activity is used to confirm proper pasteurization of skimmed or whole milk(Sharma et al., 2003). The level of ALP in milk is variable depending on the source of raw milk, and the permitable value by the U.S. and European countries is 0.35 units/L in pasteurized drinks. Thus, ALP level or activity can be employed to measure degree of pasteurization of drinks, especially milk and its derivatives(Sharma et al., 2003; Oliver et al., 2005; Klotz et al., 2008; Rankin et al., 2010; Albillos et al., 2011). Considering the importance of ALP in food safety and clinical diagnosis, many methods such as spectrophotometry(Bianchi et al., 1994), electrochemistry(Ito et al., 2000; Murata et al., 2009; Santiago et al., 2010; Miao et al., 2011; Jiang and Wang 2012; Zhang et al., 2013), chemiluminescence(Blum et al., 2001; Salter and Fitchen 2006; Albillos et al., 2011) and colorimetric assays (Sharma et al., 2003; Choi et al., 2007; Li et al., 2013) have been developed to analyze ALP. Although spectrometric method is sensitive and accurate it requires expensive instruments, professional operators, timeconsuming procedures, complicated sample processes and large amount sample, greatly limiting its applications for in-field food inspection(Bianchi et al., 1994; Thompson et al., 1991). Electrochemical and chemiluminescence methods have been explored to achieve sensitive detection of ALP(Ito et al., 2000; Murata et al., 2009; Santiago et al., 2010; Miao et al., 2011; Jiang and Wang 2012; Zhang et al., 2013). However, the former needs complicated instruments for electrochemical detection while the latter relies on desktop equipment.
Even though some of these assays have shown high sensitivity, their
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applications for in-field testing, especially for rural countries that do not have enough laboratory resources, are very difficult for distant services. Thus, a simple, portable, disposable and quantitative ALP detection tool is highly desired to fulfill daily food safety supervision, especially for under-developed regions. Recently, metal nanoparticles such as gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) have been used as signal reporters in colorimetric assays for ALP detection(Choi et al., 2007; Li et al., 2013). These methods are based on ALP induced distinguishable color changes for qualitative or semi-quantitative detections. UV-vis spectrometer has been used for quantitative analysis, but hindering point-of-care application. A reflectoquant® ALP test kit has been developed by Merck to evaluate pasteurization of milk. However, an eight-step sample handling process is needed and the detection has to be performed under a specific temperature (37°C)( Merck KGa-D64271, 2002). Obviously such a test kit is only suitable for ALP analysis in special labs. In vitro lateral flow strips based on the principle of immunochromatography have been widely employed to detect different chemical and biochemical molecules in various applications(Glynou et al., 2003; Mao et al., 2009; Xu et al., 2009; Li et al., 2010; Drygin et al., 2012; Liu et al., 2012). This method allows a one-step, rapid and inexpensive analysis by end users. Sharma et al. have developed a dry-reagent strip to examine ALP activity, which is based on ALP reaction with p-nitrophenyl phosphate in the presence of water to liberate p-nitrophenol and inorganic phosphate. Then p-nitrophenol reacts with a specific chromogen to change the color of the strip from light blue to green, which can be visualized by naked eyes(Sharma et al., 2003). This strip is a good candidate for remote
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or rural areas where expensive instruments are not available. Unfortunately, pnitrophenyl phosphate may cause eye, skin and respiratory tract irritation(Fisher Scientific 2012), while p-nitrophenol is extremely hazardous in case of ingestion and very hazardous in case of skin and eye contact as well as inhalation(Gao et al., 1997). In addition, the capability for quantitative analysis by this dry-reagent strip has not been disclosed. Herein, we report a lateral flow-through strip to detect the activity of ALP by using phosphotyrosine-capped AuNPs (AuNPs@Tyr-p) as a signal reporter. The accumulation of AuNPs at the testing zone results in a purple-brown color that can be clearly observed by naked eyes. The images are captured by a smartphone camera and are further analyzed by a home-programmed MATLAB® code to sensitively and quantitatively monitor ALP in raw cow milk. The results clearly show that the developed bio-hazards free, lateral flow-through strip is capable of portable in-field detection of ALP. 2. Materials and methods 2.1 Materials HAuCl4, sodium citrate, anti-phosphotyrosine antibody, O-Phospho-L-tyrosine, alkaline
phosphatase,
L-cysteine,
glutaraldehyde,
NaBH4,
polyethylene
glycol
(MW:20000), bovine serum albumin were purchased from Sigma Aldrich. Dialysis bag (8000-14000D) were received from scientific research special (USA). Glass fiber (SB08), polyester fiber paper (DL 42), nitrocellulose membrane (sartorius CN140), adsorbent pad (SX27) and polyvinyl chloride (PVC) backing card were ordered from Shanghai Kinbio
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Tech (China). All other chemicals used in this study are analytical grade. The deionized (DI) water used in all experiments was produced by PURELAB flex system, ELGA Corporation. 2.2 Preparation of AuNPs@Cys-Tyr-p conjugates In a typical experiment, 2 mL 1% (m/V) HAuCl4 (diluted in DI water) was mixed with 198 mL DI water in a 500 mL flask. The thoroughly mixed HAuCl 4 solution was boiled in an oil bath. Then 4 mL 1% (m/V) sodium citrate (diluted in DI water) was added into boiled HAuCl4 solution for around 30 min, when color of the solution turned to red and no further color change occurred. A two-step conjugation approach was conducted to prepare Au nanoparticles@Cysteine-phosphoTyrosine (AuNPs@Cys-Tyr-p) conjugates (Scheme1). First, 10 mL L-cysteine (1mg mL-1) was added into 10 mL AuNPs solution under continuous stirring at room temperature (RT) for 2 h. The mixture was dialyzed with DI water at 4°C for 10 h by replacing the DI water every 2 h to obtain cysteine functionalized AuNPs (AuNPs@Cys). 5 mL O-Phospho-L-tyrosine (500 μM) was mixed with 5 mL AuNPs@Cys solution. Then 100 μL glutaraldehyde solution (0.25% in DI water, v/v) was slowly dropped into O-Phospho-L-tyrosine and AuNPs@Cys mixture, followed by 1 h stirring at RT. 50 μL 0.1% (w/w) NaBH4 was added into the above mixture to reduce Schiff’s base formed between aldehyde and amine. Finally, the product was dialyzed with DI water for 10 h and then concentrated with polyethylene glycol (WM 20000) to 5 mL. The harvested phosphor-tyrosine conjugated AuNPs (AuNPs@Cys-Tyr-p) was characterized and used for lateral flow-through strip construction.
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Scheme 1.
2.3 Characterization of AuNPs@Cys-Tyr-p conjugates Morphology of AuNPs was characterized using a field emission scan electron microscope (FESEM, JSM-7800F). For FESEM analysis, the prepared AuNPs solution was drop-cast on a silicon wafer and allowed drying in open atmosphere. UV-vis spectra of
pristine
and
modified
AuNPs
were
recorded
using
Shimadzu
UV-vis
spectrophotometer (UV-2550, Japan). Zeta potential measurements were performed with Malvern Zetasizer Nano ZS 90 (Malvern, UK) equipped with a standard 633 nm laser. Binding energies were measured by X-ray photoelectron spectroscopy (XPS) (XPS, Thermo, USA) with C1s binding energy at 285.05eV as a reference. The sample for XPS characterization was prepared by placing 100 μL of the prepared solutions on a clean glass slide. 2.4 Assembling of ALP detection lateral flow-through strip The test strip consists of a sample-loaded pad, AuNPs conjugation pad, nitrocellulose membrane, adsorbent pad and backing card (Scheme 2). The sample loading pad (20 mm×4 mm) was made from a glass fiber paper and the conjugation pad (8 mm×4 mm) was made of a polyester fiber paper. The test zone was prepared by dispensing of 1.5 μL of 2 mg mL-1 anti-phosphotyrosine monoclonal antibody (anti-Tyr-p mAb) onto the nitrocellulose membrane (25 mm×4 mm). After drying at room temperature (RT) for 1 h, the membrane was blocked with 2% BSA for 30 min and then
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dried at RT. Then, all of the parts were assembled on a plastic adhesive backing card, and each part overlapped 2 mm to ensure the solution migrating through the entire strip during assays. Finally, the strip was assembled into a strip cassette for further use. 2.5 ALP detection on lateral flow-through strip The sample solution containing a desired concentration of ALP was added onto the sample pad, in which the heat-denatured ALP was used here as a negative control. After about 10 min, the test strip was imaged by a smartphone integrated digital camera (vivo S7i, 5×106 pixels). All experiments were repeated at least three times independently. 2.6 Colorimetric changed analyzed by NIH ImagJ The grey value of the testing pad was quantified by NIH free software ImageJ. For each strip, to eliminate the influence of uneven illumination, the AuNPs accumulation induced colormetric change can be quantified by taking a calculation defined below: G.I.= [(A1-A0)/A0]×100%
(1)
where G.I. is the normalized grey intensity of the testing zone, A1 is the grey value of the mAbs immobilization zone, and A0 is the grey value of neighborhood unreacted/non Ab immobilized area of the strip. Through this calculation, the more AuNPs captured by mAbs at the testing zone, the larger G.I. is obtained. 2.7 Detection strategy Scheme 2 illustrates the configuration and principle of the AuNPs based lateral flow strip. During the assay, an aqueous sample containing active ALP was dropped on
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the sample loading pad as shown in Scheme 2A (step1). Subsequently, the analyte migrates along the strip via capillary action and then reacts with AuNPs@Cys-Tyr-p in the conjugation zone. Active ALP can convert AuNPs@Cys-Tyr-p to AuNPs@Cys-Tyr by removing phosphate groups (step 2). AuNPs@Cys-Tyr cannot be captured by antiTyr-p mAb, thus no color changes can be observed at test line (step 3). A sample without ALP or with denatured ALP cannot remove the phosphate group of AuNPs@Cys-Tyr-p, which are seized by anti-Tyr-p mAb, resulting in a visual band because of the accumulation of AuNPs at test line (Scheme 2B). Therefore the ALP activity is reflected by the color changes at the testing line. In this case, the more active ALP in the sample, the less AuNPs conjugates would be captured at the test line. Sample containing active ALP will give a “white or light” line, while negative sample will give a “pink/red or dark” line. According to the principle described above, grey intensity on the test line would be reversely proportional to active ALP in samples. Scheme 2.
2.8 Statistical analysis Results were provided as means ± standard errors (SE). The data were analyzed by Student’s t-test using Origin Statistic software (Origin Lab Corporation, USA). A p-value < 0.05 was considered significant. 3. Results and discussion 3.1 Amino acid conjugated AuNPs
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Preparation of AuNPs@Cys-Tyr-p with good size-distribution and dispersion is the key factor for successfully fabrication of nanoparticle based flow-through strip. A twostep conjugation strategy as illustrated in Scheme 1 was applied to synthesize phosphotyrosine functionalized AuNPs. Table 1 summaries the reaction condition and the quality of obtained amino acid-functionalized AuNPs.
The first step is to prepare
cysteine-functionalized AuNPs (cysteine@AuNPs) by simply mix AuNPs and cysteine solution. It was found that a lower concentration of cysteine will not lead to aggregation after the reaction mixture was dialyzed against DI water (pH=6.5). This phenomena is well in-line with the reported literature (Majzik et al., 2009).
During the second
conjugation step, O-phospho-L-tyrosine is linked to cysteine@AuNPs by cross-linker glutaraldehyde. The conjugation condition was titrated to avoid high concentration of amino acid and glutaraldehyde induced particle aggregation (Migneault et al., 2004). The results show that O-phospho-L-tyrosine (500 μM, 5mL) mixed with 5 mL cysteine@AuNPs and glutaraldehyde (0.25%, 100 μL) for 1 h will no lead to AuNPs aggregation. Table 1 With the optimized condition, AuNPs@Cys-Tyr-p was synthesized for property characterization and lateral flow-through strip fabrication. Field emission scanning electronic microscopy (FESEM) result (Fig.1A) shows that the synthesized AuNPs are uniform. The UV-vis absorption spectra of the AuNPs, AuNPs@Cys, and AuNPs@CysTyr-p (Fig. 1B) reveal that the adsorption peak is narrow and close to the AuNPs’ surface plasmon resonance range (~520 nm). The adsorption peak of AuNPs is at 519 nm
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(Fig. 1A, a). Conjugation with cysteine results in a red shift of the adsorption peak to 525 nm (b). While AuNPs@Cys-Tyr-p gives an adsorption peak centered at 530 nm (c). Furthermore, pristine and modified AuNPs were examined by zeta potential measurements. The zeta-potentials of AuNPs, AuNPs@Cys, and AuNPs@Cys-Tyr-p measured in DI water (Fig. 1C) show that AuNPs@Cys-Tyr-p gives the largest negative value (-34.67 ± 1.35 mV, n=3), which should be resulted from the conjugation of negatively charged phosphate group on the tyrosine. The large potential indicates that AuNPs@Cys-Tyr-p has good dispersibility in an aqueous solution. The particle size analysis indicates that diameter of the bare AuNPs is around 16.16 ± 0.26 nm. With the conjugation of cysteine and phosphotyrosine, sizes of the nanoparticles increase to 17.19 ± 0.84 nm and 21.70 ± 0.93 nm, respectively (Fig. 1D).
Figure 1
XPS analysis in Fig. 2A shows that the typical Au 4f5/2 and Au 4f7/2 doublets with binding energies of 87.3 and 83.6eV are clearly observed, confirming the metallic state of the prepared nanoparticles. The N 1s core-level spectrum with an expected major peak at 400.1eV indicates that the amino acid is successfully attached to AuNPs (Fig. 2B). The P 2p region displays a characteristic peak at 133.2eV, which can be assigned to phosphate group of the O-Phospho-L-tyrosine (Fig. 2C). The C1s core-level spectra can be fitted into four peak components with binding energies at 285.4, 284.7, 286.9 and 288.2eV, attributing to the C-C, C=C, C-O C-S, C-N and
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, respectively (Fig. 2D).
Figure 2
The combined results from UV-vis absorbance spectra, zeta potentials and XPS characterization leads to a clear conclusion that AuNPs@Cys-Tyr-p was successfully prepared through a two-step conjugation approach, in which cysteine works as a heterobifunctional cross-linker attaches on AuNPs through the interaction of Au and –SH group, while providing–NH2 group to covalently link of phosphotyrosine. The negatively charged phosphate group on tyrosine side chain not only prohibits aggregation of AuNPs, but serves as the target of ALP. It is further proves that during the functionalization process, dialysis instead of centrifugation is successfully used to remove free amino acid for avoiding
AuNPs aggregation. Different batches of AuNPs@Cys-Tyr-p were
quantified by measuring the absorbance at 530 nm. In all remaining experiments, AuNPs@Cys-Tyr-p solution with an absorbance of ~0.27 was used to prepare the ALP lateral flow strip. 3.2 Optimization of lateral flow-through strip The amount of AuNPs@Cys-Tyr-p loaded on conjugation pad and the concentration of antibody on testing zone directly affect colorimetric response of the strip since the signal mainly depends on the amount of AuNPs@Cys-Tyr-p captured by the immobilized antibody on the test line. Different amount of AuNPs@Cys-Tyr-p conjugates and anti-phosphore-tyrosine monoclonal antibody (anti-Tyr-p mAbs) were applied in a chessboard assay (Fig.3A) to find optimal condition for a high signal-tonoise ratio. The difference of the colors in the testing zone can be distinguished by naked 12
eyes. In addition, the grey intensity of the testing zone was quantified by NIH ImageJ as described in Section 2.6. As shown in Fig. 3B, the chessboard result indicates the highest G.I. of 29.4 was resulted from the combination of dispensing 40 μL AuNPs@Cys-Tyr-p conjugates and 2 mg mL-1 anti-phosphotyrosine antibody. In following experiment, the optimal AuNPs@Cys-Tyr-p conjugates and antibody concentrations were used to fabricate ALP lateral flow-through strip. Figure 3
3.3 Smartphone based in-field detection strategy The enthusiasm of the developed AuNPs@Cys-Tyr-p based strip will be enhanced if the testing images can be analyzed by portable devices such as a smartphone. Therefore, a MATLAB® (Mathworks cop, USA) code was developed to quantify optical intensity of the testing zone on the strip. The smartphone acquired images were processed by the following procedures: a. each color image was separated into Red, Green and Blue channel, and all channels were then stacked into one grey scale image by taking a weighted sum. The stacked image was considered as a matrix of brightness values; b. For each strip, the program automatically selected a number of reference spots from unreacted area (non antibody immobilization zone) close to the testing zone (antibody immobilization zone). Reference brightness value (RefB) was calculated by taking the average of all reference spots in each strip; c. For each pixel in testing zone, its brightness signal was normalized by a calculation defined below: Brightness (Br)= (RefB-TestB)/RefB
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(2)
where TestB is the brightness value of this pixel. The effect of uneven illumination can be minimized by this normalization operation; d .Each row of the normalized matrix was considered as a curve of brightness values. These curves were aligned, stacked into one curve and then smoothed by adopting the moving-polynomial smoother to reduce the noise. Finally, the peaks of each stacked curve are automatically recorded by the program. The typical photographs of control (denatured 100 U L-1 ALP) and 100 U L-1 ALP captured by mobile phone camera were shown in Fig. 4A, clearly indicating that existence of ALP significantly reduces the pink/purple color at testing zone. The smartphone camera captured picture was quantitatively analyzed by the MATLAB® code as described above. The results show that the purple/dark band from sample with denatured ALP is characterized by a peak value of 100 (a.u.). 100 U L-1 active ALP induces a dramatic decrease of peak value (Fig.4 B). Since the MATLAB® code can be programmed to a mobile phone APP, providing an economic and portable quantitative analysis solution for the developed ALP lateral flow strip. The combination of disposable ALP lateral flow-through strip and popular smartphone highlight the potential of daily food sanitation supervision by end user.
Figure 4
3.4 The optimized condition for one-step detection of ALP by AuNPs@Cys-Tyr-p based lateral flow-through strip To achieve a high analytical performance, the assay conditions of pH and temperature were optimized. To quantify the ALP activity induced colorimetric changes,
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the assayed strips were imaged by smartphone camera. The brightness (Br) of testing zone of each strip was quantified with the strategy detailed in section 3.3 and the ALP activity induced colorimetric changes (ΔBr % ) was calculated as follow: ΔBr % = [(Brcontrol – BrALP)/Brcontrol] × 100%
(3)
where Brcontrol is the normalized brightness of control sample (denatured ALP spiked milk), BrALP is the normalized brightness of strip for measuring ALP-spiked milk. The colorimetric change (ΔBr%) is plotted as a function of different concentrations of ALP in milk. From pictures in Fig.5A, clear pink/purple bands can be observed from strips tested analyte without ALP (control, 0 U/L). Although it is documented that ALP prefers a alkaline condition, the assay buffer with a pH value of 10 does not results in good performance. While, pH 8 favors the capture and binding activity of antibody and might not inhibit the activity of ALP. Therefore, more AuNPs@Cys-Tyr-p can be seized by the anti-Tyr-p mAb and the largest colorimetric changes can be achieved by 1 U/L ALP. To evaluate the effect of temperature on the assay’s performance, the strips and samples were place at 15, 25, 37 and 45°C prior to the tests (Fig.5B). Unexpectedly, 37°C as a physiological favorable condition does not give the highest ALP induced colorimetric change. It may due to the increased non-specific interaction of the amino acid functionalized AuNPs and the mAb. Encouragingly, in terms of practical application, ambient temperature (25°C) will simplify the testing condition because incubation at 37°C or higher temperature that needs specific instrumentation will be avoided. Milk from most mammals normally contains low concentration of vitamin C and lactose, a
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sugar composed of glucose and galactose molecules. To investigate if the co-existing components may affect the performance of the AuNPs@Cys-Tyr-p based lateral flowthrough strip, ascorbic acid (5 mg/mL), lactose (10 mg/mL) and glucose (2 mg/mL) were spiked into samples containing 1U/L ALP. The quantitative ΔBr% from different anlytes are very close, indicating those biological molecules will not infer the ALP activity induced colorimetric change (Fig.5C). In summary, to reservoir the better biological activity of both antibody and the enzyme, analytes should be keep in a solution with pH value of 8. While to easy the practical application and to reduce the non-specific binding, the assay can be conducted in ambient temperature. Co-existing of vitamin C and sugar will not affect the performance of the strip, demonstrating the good specificity of the assay. With the optimized assay condition, the strip was applied for testing ALP in milk. Figure 5
3.5 Performance of AuNPs@Cys-Tyr-p based lateral flow-through strip in detecting ALP in milk To explore the feasibility of AuNPs@Cys-Tyr-p based ALP strip for milk sanitation inspection, the strip was applied to detect ALP-spiked milk. Because milk contains a large amount of macromolecule, such as protein and lipid, it is important to set up a sample pretreatment condition to achieve a sensitive detection. Centrifugation was used to pre-treat milk for reducing unexpected interference from various components and additives. Fig. 6A shows that centrifugation process (6000 rpm for 5 min at 4°C) sharply
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increases signal-to-nose ratio. It can be explained that centrifugation can assist separate and remove of lipid component which can affect the migration of AuNPs@Cys-Tyr-p on the lateral flow-through strip. Next, different concentrations of ALP were spiked into 0.1% centrifuged commercial available pasteurized milk and tested by developed strips. Fig.6B shows typical response of the strip within 10 min for ALP at concentrations of 0, 1 and 100 U L-1, respectively. Denatured ALP spiked milk was treated as a negative control. It is observed that the purple/pink color at testing zone become weaker with the increase of ALP concentration (Fig. 6B). To investigate whether the AuNPs@Cys-Tyr-p based strip could provide quantitative detection of ALP in milk, the test zones of strips were imaged by smartphone camera and the brightness (Br) of testing zone of each strip reacted with different amount of ALP was quantified with a home programmed MATLAB® code (formula 2 at section 3.3) and the ALP activity induced colorimetric change (ΔBr%) was calculated as equation (3). The quantified ΔBr % is plotted as a function of different concentrations of ALP in milk. The typical dose-signal response curve was drawn from three independent tests was shown in Fig. 6C. The detection range of the strip covers 0.1150 U L-1 (y= 38.30+21.86x, R2=0.999, n=3). Considering milk sample was diluted 1000-fold, for undiluted milk the detection limit of 0.1 U L-1 is equivalent to 100 U L-1, which is comparable to level of the ALP in raw milk (500-1100 U L-1) (Payne et al., 2009).
Figure 6
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Activity of ALP in fresh raw cow milk sample collected directly from a farm was tested by the developed ALP lateral flow-through strip. The results show that diluted (0.1%, v/v) raw milk compared to commercial pasteurized milk gives a ΔBr% of 30.27 ± 1.17 (n=3, p
