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Development of an indicator displacement based detection of malaria targeting HRP-II as biomarker for application in point-of-care settings Babina Chakma, Priyamvada Jain, Naveen K. Singh, and Pranab Goswami Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03315 • Publication Date (Web): 23 Sep 2016 Downloaded from http://pubs.acs.org on September 25, 2016
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Analytical Chemistry
Development of an indicator displacement based detection of malaria targeting HRP-II as biomarker for application in point-of-care settings Babina Chakma, Priyamvada Jain, Naveen K. Singh and Pranab Goswami*. * Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, India. ABSTRACT: A novel label free spectrophotometric detection of malarial biomarker HRP-II following an indicator displacement assay has been developed. The assay is based on competitive displacement of murexide dye from its complex with Ni 2+ by HRP-II present in serum samples. The binding constant (Kd) discerned for the dye and HRP-II to Ni2+ were 1.4 x 10-6 M-1 and 6.8 x 10-9 M-1, respectively. The progress of the reaction could be monitored from the change of color from orange (~λ482 nm) to pink (~λ515 nm) with the concomitant increase in HRP-II concentration in the mixture. A linear response (R 2 =0.995) curve was generated by plotting the ratio of absorbance (λ515 nm/λ482 nm) against the HRP-II concentrations. The method offers to detect HRP-II as low as 1 pM without any interference from some common salts and the major protein, HSA present in the blood serum. The detection method was reproduced in a microfluidic paper based analytical device (μPAD), fabricated by printing hydrophobic alkyl ketene dimer on a chromatographic paper to create hydrophilic microchannels, test zone, and sample application zone. The device offers to use a maximum sample volume of 20 ± 0.06 μl and detects HRP-II within 5 minutes with LOD of 30 ± 9.6 nM in a dynamic range of 10 to 100 nM. The method has thus immense potential to develop as rapid, selective, simple, portable and inexpensive malarial diagnostic device for point-of-care and low resource setting applications.
INTRODUCTION Malaria is a life-threatening disease occurring predominantly in the tropical regions of developing and underdeveloped countries. Weak economy and poor healthcare networks are the major hindrances in proper monitoring and eradicating malaria in these countries.1 Accurate and timely diagnosis of malaria is important for its effective remedial treatment which demands an affordable, stable, simple, sensitive and fast malaria diagnostic tool or technique. Presently, Rapid diagnostic tests (RDT), which are based on antibody as recognition element, have been widely used in these malaria prevalent regions owing to their many positive qualities such as, simplicity to operate, they are fast and are inexpensive.2 However, there are reports indicating several issues on these antibody based RDTs such as cross reactivity with auto-antibodies,3 low stability in hot and humid climate which leads to test failures, 4–6 false negative results during severe malaria due to prozone effect,7,8 and are non-quantitative in nature. Efforts have been made to overcome these problems by replacing antibody with other recognition elements targeting HRP (Histidine rich protein) II, a specific biomarker of P.falciparum malaria. HRP-II is a small, 30 kDa soluble protein with many tandem repeats of Ala-His-His-Ala-Ala-Asp uniquely synthesized and secreted by P.falciparum.1 The protein, involved in heme binding and heme detoxification by formation of hemozoin,9 is released in high quantity in the serum. Histidine targeted spectrophotometric sensor using Ni (II) NTA (Nickel nitrilotriacetic acid)-functionalized Au and Ag nano-
particles has been reported (Swartz et al., 2011).10 Later on using the same principle a detection system for low resource setting was developed where Ni-NTA functionalized glass slides and magnetic nanoparticles generated visible coffee ring like structures following addition of the HRP-II protein.11 They used Ni-NTA gold nanoparticles with different spacer molecules to enhance the sensitivity of HRP-II detection.12 An iridium (III) luminescent probe which selectively binds to the histidine molecules of HRP-II and gives a phosphorescent signal was also proposed.13 The sensitivity and specificity of these methods however, needs to be adequately addressed to realize their practical applicability. Additionally, these detection systems are complex and expensive due to their use of nanomaterials that require functionalization and coating for stabilization. All the aforementioned alternative methods are in conceptual stage and yet to be validated for their practical utility. Here, we developed a sensitive, simple and stable method for quantification of HRP-II using indicator displacement assay (IDA). The assay is expected to offer many advantages over the conventional assays as the IDA does not require the indicator to be covalently attached to the receptor, it works well in aqueous and organic medium as well, and can easily be tailored to different receptors and ligands.14,15A commonly available complexometric indicator dye murexide, also known as ammonium purpurate, has been used in this assay. The dye has high affinity towards nickel ions and gives an orange color upon binding to the ions. It is a hydrophilic tridentate ligand which binds to Ni2+ with three coordination bonds. HRP-II competes with the dye,
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Analytical Chemistry
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displacing Ni2+ from the murexide-Ni2+complex thus regenerating the original color of the dye. To make this sensing method economical and viable for low resource settings we replicated the detection in a microfluidic paper based analytical device (μPAD), which complies with the ASSURED (accurate, sensitive, specific, user friendly, rapid and robust, equipment free and deliverable to end users) criteria prescribed by WHO.16 The microfluidic channels on the paper surface were created by forming hydrophobic barriers using alkyl ketene dimer and inkjet printing in a very hassle free and cost effective manner.17,18 The performance of the μPAD was evaluated for detection of HRP-II and reported here.
EXPERIMENTAL SECTION Chemicals and reagents Murexide, nickel (II) sulphate heptahydrate and HEPES were obtained from HIMEDIA. Whatman filter paper grade 1 (7 X 10 mm) and Human serum albumin (HSA) were purchased from Sigma. Alkyl Ketene Dimer dimer 1840 (AKD) purchased from Flourish paper & chemicals Limited (Mumbai, India). All the other reagents used were of analytical grade. All solutions were prepared in ultrapure water with resistivity >18 MΩ. The experiments were performed at room temperature (RT) unless stated specifically. Cloning, Expression and Purification of HRP-II A pET 3D vector containing the HRP-II sequence was obtained from BEI resources (USA) and transformed into BL21 cells. Protein expression was performed at an optimized concentration of 0.5 mM IPTG at 28 ˚ C for 12 hrs and purified to >98 % by Ni-NTA affinity column.19,20 The purity of HRP-II was further confirmed by SDS-PAGE (S1). The purified protein was then dialyzed in 20 mM HEPES buffer, pH 7.4.The concentration of the purified protein was ~ 0.27 mg/ml (biuret assay). Spectrophotometric studies The IDA reactions were performed in 20 mM HEPES buffer at pH 7.4. The reaction mixtures were allowed to incubate at RT for 10 mins, absorbance was taken at λ515 and λ482 nm in a flat bottom 96 well microtiter plate in a microtiter plate reader (TECAN M200 Pro, Switzerland). Spectral characterization was performed by UV –Vis spectrophotometer (Cary 200 Bio, Agilent). Graphs were plotted using Sigma Plot software and the data points plotted in the graph were the mean of three measurements, while the error bars represent the relative standard deviation. Preparation of real serum Real serum samples were collected from healthy volunteers following an established protocol and ethical guideline. In an anticoagulant free collection tube, 12 ml of blood was collected and allowed to settle for 15-30 mins to clot. The samples were then centrifuged at 2000 g for 10 mins in a refrigerated tube to remove the clot. The supernatant collected was designated as serum and was used for experiments. Fabrication of paper based μPAD
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μPAD was developed on Whatman filter paper 1. The design patterns were created by using Adobe illustrator CS6. The pattern consisted of a circular sample zone of 8 mm (dia) and 6 test zones of 4 mm (dia) for enabling to repeat six measurements. The sample zone and the test zones were connected by channels which had a dimension of 2 mm (W) x 4 mm (L) and thickness of 180 μm. The AKD printing was done in a HP deskjet-100 printer. The cartridge of the printer was modified by replacing the hydrophobic foam with hydrophilic chromatography paper and absorbent cotton. The ink cartridge was filled with 0.5% (w/v) AKD solution in n-heptane. After the printing, the paper was heated on a hot plate at 100˚C for 10 min and then allowed to cure for few hrs before use (S2). The AKD printed paper was characterized by scanning electron microscopy (SEM) (LEO 1430 VP, ZEISS, Germany). The samples were gold coated for 120 s at 5 mA and were mounted on the sample holder using carbon tape. The samples were examined under low beam energies (5-8 EHT) at RT. Atomic force microscopy (AFM) using ambient air scanning probe microscope (Agilent Technologies 5500,USA) was also used to check the topography and roughness of the paper substrate before and after the printing. The images were taken with tapping mode using picoscan 5 software. Detection of HRP-II on μPAD The samples migrated to the test zone were allowed to react with the reagents at ambient conditions. After the reaction the paper platforms were allowed to dry for 15 mins. The images of the color developed on the paper platform following the sample applications were acquired by using HP scanner. The images taken by HP Laser jet pro M1132 MFP were transferred to a computer, converting them to a CMYK mode to quantify the response using Adobe Photoshop (CS6). The magenta filter was considered to determine the pixel intensities.21 The mean pixel intensity corresponding to the analyte concentration was calculated by the histogram feature. All data points presented here are the mean intensities of six measurements against each concentration using independent devices, while the error bars represent the relative standard deviation.
RESULTS AND DISCUSSION. Spectrophotometric detection of HRP-II Free murexide solution exhibited an intense peak at ~λ515 nm. The peak shifted to ~λ482 nm when Ni2+ solution was added to it (Fig 1A). For optimization of the reaction, 50 µM of murexide was titrated with increasing concentrations of Ni2+. The saturation was reached at ~50 µM of Ni2+ Fig 1 B), indicating 1:1 stoichiometry of the reaction. From the plot the binding constant (Kd) of 1.4 X 10-6 M-1was discerned for the dye from the equation Y=BmaxX/Kd+X, where Bmax is the maximum available concentration of receptors, X is concentration of ligand and Y is concentration of receptor. The ratio metric (515 nm/482 nm) response was considered to improve the reliability of the assay. 22 An equimolar (50 µM) mixture of murexide and Ni2+ when titrated with HRP-II, the orange color (~λ482 nm) of the solution gradually changes to pink (~λ515 nm) with the corresponding increase in protein concentration (Bottom panel in Fig 2). Using the said concentrations of dye and Ni2+, a dynamic detection range of 1 nM –3 µM and a limit of detection (LOD) of 53± 7.1 nM for HRP-II (R2 =0.995) were discerned using LOD =
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Analytical Chemistry
3*Sy/b, where Sy is the standard deviation (SD) of y-intercept, and b is the slope of the linear curve. The method could detect as low as 1 pM of HRP-II in the solution. The Kd value calculated for HRP-II-Ni2+ was 6.8 x10-9 M-1. This higher Kd value of HRP-II than the murexide dye for Ni2+ validates the ability of HRP-II to displace Ni2+ easily from the dye complex. Notably, the high affinity binding of Ni2+ to His amino acid is commonly
exploited to purify His-tag proteins and HRP-II through column chromatographic technique.20However, this displacement based reaction is not known to utilize for detection of HRP-II and other proteins. The method offers a fast, sensitive and label free detection of HRP-II. Moreover, the analysis does not involve any expensive reagents hence it has great potential to use in a resource limiting platform.
Figure 1. (A) Change of UV-Vis spectra of murexide dye upon addition of increasing concentration of Ni2+ and (B) Absorbance ratio of A515/ A482 for the reaction of murexide dye with the increasing concentration of Ni 2+ in 20 mM HEPES buffer pH 7.4.
Figure 2: Plot of absorbance ratio of A515/ A482 for a fixed amount of murexide-Ni2+ (1:1) against increasing concentration of HRP-II in 20 mM HEPES buffer pH 7.4. The linear region of the data (shown in inset A) was fit to a linear equation. The cluster of data in picomolar range was expanded (shown in inset B) for clarity. Panel below the graph is a real representative image of HRP-II detection in solution.
Effect of pH on spectrophotometric detection of HRP-II The effect of pH on the displacement reaction was investigated (Fig 3 A). The response was conspicuous in alkaline conditions, drastically increased with increasing pH value from 7 to 9 (inset in fig 3 A). At lower pH (
