- Email: [email protected]
Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor
Accepted Manuscript Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor
Neelam Verma, Ashish Kumar Singh, Nancy Saini PII: DOI: Reference:
S2214-1804(17)30001-6 doi: 10.1016/j.sbsr.2017.07.004 SBSR 201
To appear in:
Sensing and Bio-Sensing Research
Received date: Revised date: Accepted date:
17 February 2017 20 July 2017 31 July 2017
Please cite this article as: Neelam Verma, Ashish Kumar Singh, Nancy Saini , Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor, Sensing and Bio-Sensing Research (2017), doi: 10.1016/j.sbsr.2017.07.004
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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor Neelam Verma*, Ashish Kumar Singh and Nancy Saini Biosensor Technology Lab, Department of Biotechnology, Punjabi University, Patiala-147002,
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India
*Dr. Neelam Verma
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Professor
Department of Biotechnology
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Punjabi University Patiala
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Email: [email protected]
ACCEPTED MANUSCRIPT Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor
Abstract
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Arginine deiminase co-immobilized with ZnS QDs coupled micro-disk was applied for the
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sensing of arginine in real and spiked fruit samples. Intracellular arginine deiminase from Lactococcus lactis MTCC 460 was partially purified by ammonium sulphate precipitation and
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co-immobilized on hydrosol gel disk with ZnS quantum dots. Surface study and topology of immobilized ZnS QDs were characterized by SEM. The size of MPA capped ZnS quantum dots
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was achieved up to 200 nm. Excitation and emission wavelength of synthesized ZnS QDs was observed to be 259 and 580 nm respectively. Linear range of detection of arginine was found to
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be 1.0 to 10-4M and developed biosensor was used to monitor arginine in water melon and pomegranate fruit juices. Main advantage of the developed system is there is no need of
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pretreatment of sample for the estimation of arginine content.
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Keywords: Arginine, phosphorescence, Quantum dots, hydrosol gel, biosensor etc.
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1. Introduction
Quantum dots (QDs) a nanorystals semiconductor with light-emitting property has been recently
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popular for use as biomolecular detection tool because of their distinctive optical properties, and has advantages over traditional fluorophores like many organic dyes [1,2]. QDs utilize wide
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range of applications like live cell imaging, fixed cell and tissue labeling and high throuput biosensors. QDs have made them excellent donors for fluorescence resonance energy transfer (FRET) sensors due to its narrow sized and symmetric emission spectra. Additionally, the overlap between the emission spectra of the donor and acceptor is reduced, and the interaction is bypassed in FRET pairs [1-3]. The broad excitation spectra of QDs facilitate excitation at a single wavelength far removed (>100 nm) from their respective emissions, allowing QDs to be used in multiplex assays with single excitation sources. the surface modification of QDs with antibodies, aptamers, and peptides with covalent or non-covalent linking approaches are the most established and widespread detection bioprobes. The long-term photostability, superior
ACCEPTED MANUSCRIPT brightness, and good chemical stability of QDs enable them to greatly improve bioassay sensitivities and limits [3-5]. Arginine is α-amino acid and abundantly found in protamines and histone proteins. It was first isolated from lupin seedlings by Hedin [6]. In mammals, arginine is classified as semiessential or conditionally essential amino acids, depending upon the growth of the individual. L-
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arginine is a great interest in life science and its metabolic derivatives and also important to
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storage, transport and excretion of nitrogen and in regulating protein metabolism in the body.
concentration of arginine available in the blood stream [7].
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Arginine biosensor can also be employed for the diagnosis of cancer by determining the
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Arginine can be detected by spectrophotometry [8], HPLC [9] and so on. However, these all methods are having their own advantages and disadvantages time-consuming, toxic and
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requiring complicating pretreatment, which hamper their further applications. Biosensors may overcome some of these problems, beause of quick response and high sensitivity. For arginine
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detection, biosensors based on potentiometry and amperometry were previousaly developed and reported [10-14,36].
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Previous studies on QDs based biosensors were focused on fluorescence phenomenone and negative points of these processes are short-lived autofluorescence and scattering light from 4
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biological matrices that led to interference and noise in signaling process [34,35]. Recently, the T1(4G)-6A1(6S) transition of Mn2+ in Mn-doped ZnS QDs utilizes into room-temperature
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phosphorescence (RTP) detection has attracted much attention, and is widely studied for developing sensors with great success, and has become a hotspot [15-25]. Employing this
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approach, the interferences from biological matrices can be avoided by phosphorescent QDs because the long-lived phosphorescence has a suitable delay time. The selectivity is also enhanced because phosphorescence is less common than fluorescence. Reportedly, Mn-doped ZnS QDs exhibit promising phosphorescence emission (̴ 580 nm), which is produced by the energy transfer from the band gap of ZnS to Mn2+ dopant and the subsequent transition from the triplet state (4T1) to the ground state (6A1) of the Mn2+ involved in the ZnS host lattice [26]. The utilization of phosphorescent QDs in optical sensing is still at the initial stage but has been proven to be very prospective [33].
ACCEPTED MANUSCRIPT In this paper, a simple scheme has been proposed to prepare the arginine-sensing system,
which is composed of water-soluble Mn-doped ZnS phosphorescent QDs
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enzyme arginine diaminase. That allows effective and quantitative detection of arginine. Colloidal Mn2+-doped ZnS nanoparticles showing RTP emission were synthesized and then made water-soluble by capping mercaptopropionic acid (MPA) onto the surface of QDs. Such coating of the nanoparticles did not change their emission properties [32], but were
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effective for quantitative detection of arginine.
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2. Experimental Section 2.1.Materials and Chemicals
Arginine, Zinc Acetate, Sodium sulphide, Magnisium acetate, sodium hydroxide, 3-mercapto
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propanoic acid (3-MPA), Sodium phosphate (monobasic) and potassium phosphate (dibasic) were procured from Himedia ltd. (Mumbai, India). Enzyme arginine deiminase was partially
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purified from Bacteria Lactococcus lactis MTCC460. Ultrapure water (18.2 MΩ cm) was obtained from a WaterPro water purification system (Labconco Corporation, Kansas City, MO).
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Fruit juice samples was procured from the local market. 2.2.Apparatus
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The morphology and microstructure of the QDs were characterized by Scanning Electron microscopy (SEM, JEOL, JSM-6010LV). The phosphorescence measurements were performed
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on fibre optic spectrophotometer (Maya2000, Oceanic Optics, USA). Absorption spectra were also recorded on fibre optic spectrophotometer (Maya2000, Oceanic Optics, USA).
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2.3.Synthesis and characterization of the Mn-Doped ZnS QDs Synthesis of Mn-Doped ZnS QDs was carried out in aqueous solution based on a
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published method with minor modification [27]. Briefly, 10 mL of 0.1 M Zn(Ac), 4 mL of 0.01 M Mn(Ac), and 100 mL of 0.04 M MPA were added to a three-neck flask. The solution was mixed and adjusted to pH 11 with 1 M NaOH. After air was removed with nitrogen purging for 30 min at room temperature, 10 mL of 0.1 M Na2S was immediately injected into the solution. After stirring for 20 min, the solution was aged at 50˚C under open air for 2 h to form MPA-capped Mn-doped ZnS QDs. The QDs were purified by precipitation with ethanol and separated by centrifugation, washed with ethanol, and dried in vacuum.
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obtained QDs powder was highly soluble in water. The characterization of prepared ZnS QDs was done by Scanning Electron Microscopy (SEM) and UV-vis spectroscopy.
ACCEPTED MANUSCRIPT 2.4.Assay condition Buffers with varying basicity (pH 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11) were prepared by adding different volume of NaOH (0.1M) to a phosphate buffer solution (PBS, pH=8.0, 20mM). Prepared QDs powder was dissolved in ultrapure water to the concentration of 5mg/100µl and then 80µl of the QDs solution was added to each of the above sample and phosphorescence was measured.
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2.5.Optimization of QDs concentration for sensing disc
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Different concentration of ZnS QDs from stock solution (5mg/100µl) was diluted further in to 5,
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10, and 100 times and 20µl of each diluted ZnS QDs solution was used for the immobilization on to the disc. Concentration range of QDs was optimizing to get maximum sensing response.
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2.6.Preparation of sensing disc
Sensing disc was fabricated according to Verma et al. [28,37,38] with minor modification where,
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10µl prepared sol gel solution poured on to the transparent plastic disc and kept for gelation. The TMOS-sol gel coated disc was placed on to the top of the fiber-optic probe. Arginine solution of
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different concentration ranging from 1.0 -10-5M were prepared in 20mM phosphate buffer (pH=8). 10µl of arginine solution/sample solution was poured on to the prepared sensing disc
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and phosphorescence intensity was recorded for each individual concentration of arginine, and standard refrence chart was prepared using different concentration of arginine.
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2.7.Application of the Developed Biosensor The standard reference chart was used for quantifying arginine concentration in various fruit
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samples. Fruit samples such as orange, water melon and guava juices were taken in separate glass cells. Reliability of the developed biosensor was checked with the spiked sample by
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standard addition method. In which samples were subjected to spiked with known concentration of arginine (10mM).
3.Results and Discussion 3.1.Characterization of the Mn-Doped ZnS QDs The size of MPA-capped Mn-doped ZnS QDs was estimated by SEM analysis at different resolution. The size of the ZnS QDs was found to be around 200 to 1000 nm shown in Figure 1(A and B). Confirmation of MPA-capped Mn-doped ZnS QDs were checked by exposing in UV light (Figure 2). An eppendorf tube filled with aqueous solution of QDs at the concentration
ACCEPTED MANUSCRIPT of 5mg/L has shown fluorescence after exposing in UV light (B) as compared with another tube (A) having without QDs distilled water. Absorption spectra of the prepared ZnS QDs were performed by scanning the wavelength from 200-700 nm (Fig. 3A). The maximum absorbance peak was found at the wavelength of 259 nm. Moreover, two other peaks were also observed at 280 and 325 nm, which indicates the dispersity of QDs in solution is not the narrow size distribution [29].
As effective mass model has suggested that the absorption band of the
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quantum dots show dependency with radius of particles [30]. Fig. 3B illustrates the
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phosphorescence property of the ZnS QDs in 20mM PBS (pH 8.0). A strong phosphorescence
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peak centered at around 580 nm is observed. It also indicates the monodispersity of QDs in the buffer solution. Lin and his co-worker have also found the emission peak at 590 nm of ZnS QDs
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at room temperature [29]. 3.2.Effect of pH on Mn-doped ZnS QDs
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The phosphorescence intensity of QDs was noted in PBS at different pH ranging from 8 to 11. Table 1 shows that the phosphoresce behavior of ZnS QDs is pH dependent and has enhanced
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with increasing pH. The maximum phosphorescence intensity was found at pH 10. As earlier work also revealed that as pH is elevated the RTP also increases [29]. The chemistry behind this
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is basically deprotonation of carboxyl group because of increase in pH led to doping of Zn(II) occured and create the surface defect that ultimately protects from quencher like dissolve oxygen
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molecules [30]. Second, is the enhancment of coordination between sulfydril and metal ion group with incrase in pH and also lower the process of nonradiative transition, thereby
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enhacement in intensity was observed [27]. 3.3.Optimization of QDs concentration
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As the data shown in Table 2, The best concentration of QDs was found 5000ppm means 10 times diluted from the stock solution of 50,000ppm. Massive concentration of ZnS QDs in sol gel matrices cause agrragation of the particles and the gel becomes cloudy. Due to this formation of bigger nanoparticles are tooke place and resulted loss of phosphorescence intensity [31]. 3.4.Immobilization of enzyme with ZnS QDs Crude enzyme arginine deiminase was co-immobilized with ZnS QDs in the hydrosol gel. SEM analysis of the immobilized disk with enzyme and ZnS QDs was shown in figure 1(C and D). Figure 1C shows that dispersed ZnS QDs in hydrosol gel matrix in regular way and estimated size was found 100 to 500 nm. Different concentrations of standard arginine solution were
ACCEPTED MANUSCRIPT prepared in PBS (pH 8; 0.20 mM). The phosphorescence intensity was measured 545, 580 and 680 nm. As shown in Figure 4, the concentration of arginine decreased the phosphorescence intensity was also decreased due to decrease in pH. A standard chart was prepared using different concentrations of arginine as shown in table 3. Further it is used to estimate arginine concentration of unkown samples. The storage stability of sensing disk was also checked and it is found to be stable around two weeks without significant decreased in performance (table 3).
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3.5.Arginine detection and application in real samples
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The developed biosensor was applied for the detection of arginine with known concentrations of
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arginine 0.01 and 100mM in PBS, and standard chart was prepared (Table 3). The response time of the developed biosensor for arginine detection was found to be 5 min. further, the developed
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biosensor was applied to check arginine in fruit juices samples. The observed result showed that arginine cotents the order of watermelon >guava >pomegranate >orange juice samples. The
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reliability of the system was also checked by using simulated fruit juices and it was observed >90% reliabile. The storage stability of the biosensor was achieved one week, after that there is
4.Conclusion and future perspectives
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decreased activity by 5%.
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The maximum enzyme activity of partially purified enzyme was found to be 42.66±2 IU/ml (specific activity 8.7±0.3 IU/mg). UV-vis spectra of ZnS QDs were observed at 259 to 231 nm.
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Phosphorescence spectra peak of ZnS QDs was observed at 580 nm. Size of the ZnS QDs was found to be in the range of 200 to 1000 nm. ZnS QDs was detected at pH ranges of 8 to 11 and
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phosphorescence intensity was found best at pH 10. The phosphorescence intensity increased with increase in concentration of arginine and pH level confirm the scanning principle. Linear
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range of detection of arginine was found to be 1.0 to 10-4M and developed biosensor was used to monitor arginine in guava, orange, water melon and pomegranate fruit juices. The reliability of developed biosensor was checked with spiked fruit juices samples. The storage stability of device was found to be one week. A novel combination of ZnS QDs with partially purified arginine deiminase modified sensor disk was developed with significant feature for an on the spot biosensor: simplicity, selectivity and low cost in fabrication tgether mak it very usefull approach. Acknowledgment: The authors are grateful to the Department of Biotechnology, Punjabi University Patiala for providing facilities to carry out this work.
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Table 1: Effect of pH on Mn-doped ZnS QDs
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Phosphorescence intensity (AU) 655.61 nm
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539.26
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581.21 nm
Phosphorescence intensity at 580 nm (AU) 1.35 ±0.60 201.43 ±3.85 254.72 ±2.90 28.45 ±4.6
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ZnS QDs concentration for immobilization (ppm) 50,000 10,000 5,000 500 n=3
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Table 2: Phosphorescence intensity of different QDs concentration
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Table 3: Standard chart for arginine with storage stability Arginine concentration (M) 1 10-1 10-2 10-3 10-4 10-5
Phosphoresce intensity at 580nm (AU) 1 week 2nd week 3rd week 235.66 230.11 201.76 210.09 209.38 186.19 187.36 177.10 141.35 156.98 146.45 120.48 124.36 122.95 70.38 112.87 107.71 30.21 st
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Guava Pomegranate Watermelon Orange n=3
180.74 ±2.6 177.28 ±3.1 185.25 ±2.9 168.55 ±4.2
Spiked with 10mM arginine at 580nM 190.86 ±3.6 188.98 ±2.8 197.98 ±3.4 182.30 ±3.7
Concentration of arginine estimated
% reliability
10.16 10.08 10.56 9.72
98.4 99.2 94.6 97.2
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Fruit samples
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Fig.1: SEM analysis of ZnS QDs (A) ZnS QDs in PBS, (B) ZnS QDs in Sol gel, (C) high magnified image of ZnS QDs in Sol gel and (D) immobilized ZnS QDs with Arginine deiminase.
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Fig. 2: ZnS QDs in UV light (A): tube filled with water and (B): aqueous solution of ZnS QDs
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Fig. 3: (A) Absorbance, (B) Emission spectra of Mn-doped ZnS QDs. Solution were prepared in PBS (20mM pH 8.0).
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100 mM
10 micromole
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Fig.4. Phosphorescence response of different concentration of arginine (0.01 – 100mM).
Neelam Verma, Ashish Kumar Singh, Nancy Saini PII: DOI: Reference:
S2214-1804(17)30001-6 doi: 10.1016/j.sbsr.2017.07.004 SBSR 201
To appear in:
Sensing and Bio-Sensing Research
Received date: Revised date: Accepted date:
17 February 2017 20 July 2017 31 July 2017
Please cite this article as: Neelam Verma, Ashish Kumar Singh, Nancy Saini , Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor, Sensing and Bio-Sensing Research (2017), doi: 10.1016/j.sbsr.2017.07.004
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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor Neelam Verma*, Ashish Kumar Singh and Nancy Saini Biosensor Technology Lab, Department of Biotechnology, Punjabi University, Patiala-147002,
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India
*Dr. Neelam Verma
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Professor
Department of Biotechnology
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Punjabi University Patiala
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Email: [email protected]
ACCEPTED MANUSCRIPT Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor
Abstract
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Arginine deiminase co-immobilized with ZnS QDs coupled micro-disk was applied for the
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sensing of arginine in real and spiked fruit samples. Intracellular arginine deiminase from Lactococcus lactis MTCC 460 was partially purified by ammonium sulphate precipitation and
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co-immobilized on hydrosol gel disk with ZnS quantum dots. Surface study and topology of immobilized ZnS QDs were characterized by SEM. The size of MPA capped ZnS quantum dots
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was achieved up to 200 nm. Excitation and emission wavelength of synthesized ZnS QDs was observed to be 259 and 580 nm respectively. Linear range of detection of arginine was found to
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be 1.0 to 10-4M and developed biosensor was used to monitor arginine in water melon and pomegranate fruit juices. Main advantage of the developed system is there is no need of
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pretreatment of sample for the estimation of arginine content.
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Keywords: Arginine, phosphorescence, Quantum dots, hydrosol gel, biosensor etc.
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1. Introduction
Quantum dots (QDs) a nanorystals semiconductor with light-emitting property has been recently
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popular for use as biomolecular detection tool because of their distinctive optical properties, and has advantages over traditional fluorophores like many organic dyes [1,2]. QDs utilize wide
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range of applications like live cell imaging, fixed cell and tissue labeling and high throuput biosensors. QDs have made them excellent donors for fluorescence resonance energy transfer (FRET) sensors due to its narrow sized and symmetric emission spectra. Additionally, the overlap between the emission spectra of the donor and acceptor is reduced, and the interaction is bypassed in FRET pairs [1-3]. The broad excitation spectra of QDs facilitate excitation at a single wavelength far removed (>100 nm) from their respective emissions, allowing QDs to be used in multiplex assays with single excitation sources. the surface modification of QDs with antibodies, aptamers, and peptides with covalent or non-covalent linking approaches are the most established and widespread detection bioprobes. The long-term photostability, superior
ACCEPTED MANUSCRIPT brightness, and good chemical stability of QDs enable them to greatly improve bioassay sensitivities and limits [3-5]. Arginine is α-amino acid and abundantly found in protamines and histone proteins. It was first isolated from lupin seedlings by Hedin [6]. In mammals, arginine is classified as semiessential or conditionally essential amino acids, depending upon the growth of the individual. L-
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arginine is a great interest in life science and its metabolic derivatives and also important to
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storage, transport and excretion of nitrogen and in regulating protein metabolism in the body.
concentration of arginine available in the blood stream [7].
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Arginine biosensor can also be employed for the diagnosis of cancer by determining the
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Arginine can be detected by spectrophotometry [8], HPLC [9] and so on. However, these all methods are having their own advantages and disadvantages time-consuming, toxic and
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requiring complicating pretreatment, which hamper their further applications. Biosensors may overcome some of these problems, beause of quick response and high sensitivity. For arginine
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detection, biosensors based on potentiometry and amperometry were previousaly developed and reported [10-14,36].
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Previous studies on QDs based biosensors were focused on fluorescence phenomenone and negative points of these processes are short-lived autofluorescence and scattering light from 4
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biological matrices that led to interference and noise in signaling process [34,35]. Recently, the T1(4G)-6A1(6S) transition of Mn2+ in Mn-doped ZnS QDs utilizes into room-temperature
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phosphorescence (RTP) detection has attracted much attention, and is widely studied for developing sensors with great success, and has become a hotspot [15-25]. Employing this
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approach, the interferences from biological matrices can be avoided by phosphorescent QDs because the long-lived phosphorescence has a suitable delay time. The selectivity is also enhanced because phosphorescence is less common than fluorescence. Reportedly, Mn-doped ZnS QDs exhibit promising phosphorescence emission (̴ 580 nm), which is produced by the energy transfer from the band gap of ZnS to Mn2+ dopant and the subsequent transition from the triplet state (4T1) to the ground state (6A1) of the Mn2+ involved in the ZnS host lattice [26]. The utilization of phosphorescent QDs in optical sensing is still at the initial stage but has been proven to be very prospective [33].
ACCEPTED MANUSCRIPT In this paper, a simple scheme has been proposed to prepare the arginine-sensing system,
which is composed of water-soluble Mn-doped ZnS phosphorescent QDs
and
enzyme arginine diaminase. That allows effective and quantitative detection of arginine. Colloidal Mn2+-doped ZnS nanoparticles showing RTP emission were synthesized and then made water-soluble by capping mercaptopropionic acid (MPA) onto the surface of QDs. Such coating of the nanoparticles did not change their emission properties [32], but were
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effective for quantitative detection of arginine.
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2. Experimental Section 2.1.Materials and Chemicals
Arginine, Zinc Acetate, Sodium sulphide, Magnisium acetate, sodium hydroxide, 3-mercapto
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propanoic acid (3-MPA), Sodium phosphate (monobasic) and potassium phosphate (dibasic) were procured from Himedia ltd. (Mumbai, India). Enzyme arginine deiminase was partially
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purified from Bacteria Lactococcus lactis MTCC460. Ultrapure water (18.2 MΩ cm) was obtained from a WaterPro water purification system (Labconco Corporation, Kansas City, MO).
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Fruit juice samples was procured from the local market. 2.2.Apparatus
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The morphology and microstructure of the QDs were characterized by Scanning Electron microscopy (SEM, JEOL, JSM-6010LV). The phosphorescence measurements were performed
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on fibre optic spectrophotometer (Maya2000, Oceanic Optics, USA). Absorption spectra were also recorded on fibre optic spectrophotometer (Maya2000, Oceanic Optics, USA).
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2.3.Synthesis and characterization of the Mn-Doped ZnS QDs Synthesis of Mn-Doped ZnS QDs was carried out in aqueous solution based on a
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published method with minor modification [27]. Briefly, 10 mL of 0.1 M Zn(Ac), 4 mL of 0.01 M Mn(Ac), and 100 mL of 0.04 M MPA were added to a three-neck flask. The solution was mixed and adjusted to pH 11 with 1 M NaOH. After air was removed with nitrogen purging for 30 min at room temperature, 10 mL of 0.1 M Na2S was immediately injected into the solution. After stirring for 20 min, the solution was aged at 50˚C under open air for 2 h to form MPA-capped Mn-doped ZnS QDs. The QDs were purified by precipitation with ethanol and separated by centrifugation, washed with ethanol, and dried in vacuum.
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obtained QDs powder was highly soluble in water. The characterization of prepared ZnS QDs was done by Scanning Electron Microscopy (SEM) and UV-vis spectroscopy.
ACCEPTED MANUSCRIPT 2.4.Assay condition Buffers with varying basicity (pH 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11) were prepared by adding different volume of NaOH (0.1M) to a phosphate buffer solution (PBS, pH=8.0, 20mM). Prepared QDs powder was dissolved in ultrapure water to the concentration of 5mg/100µl and then 80µl of the QDs solution was added to each of the above sample and phosphorescence was measured.
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2.5.Optimization of QDs concentration for sensing disc
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Different concentration of ZnS QDs from stock solution (5mg/100µl) was diluted further in to 5,
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10, and 100 times and 20µl of each diluted ZnS QDs solution was used for the immobilization on to the disc. Concentration range of QDs was optimizing to get maximum sensing response.
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2.6.Preparation of sensing disc
Sensing disc was fabricated according to Verma et al. [28,37,38] with minor modification where,
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10µl prepared sol gel solution poured on to the transparent plastic disc and kept for gelation. The TMOS-sol gel coated disc was placed on to the top of the fiber-optic probe. Arginine solution of
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different concentration ranging from 1.0 -10-5M were prepared in 20mM phosphate buffer (pH=8). 10µl of arginine solution/sample solution was poured on to the prepared sensing disc
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and phosphorescence intensity was recorded for each individual concentration of arginine, and standard refrence chart was prepared using different concentration of arginine.
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2.7.Application of the Developed Biosensor The standard reference chart was used for quantifying arginine concentration in various fruit
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samples. Fruit samples such as orange, water melon and guava juices were taken in separate glass cells. Reliability of the developed biosensor was checked with the spiked sample by
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standard addition method. In which samples were subjected to spiked with known concentration of arginine (10mM).
3.Results and Discussion 3.1.Characterization of the Mn-Doped ZnS QDs The size of MPA-capped Mn-doped ZnS QDs was estimated by SEM analysis at different resolution. The size of the ZnS QDs was found to be around 200 to 1000 nm shown in Figure 1(A and B). Confirmation of MPA-capped Mn-doped ZnS QDs were checked by exposing in UV light (Figure 2). An eppendorf tube filled with aqueous solution of QDs at the concentration
ACCEPTED MANUSCRIPT of 5mg/L has shown fluorescence after exposing in UV light (B) as compared with another tube (A) having without QDs distilled water. Absorption spectra of the prepared ZnS QDs were performed by scanning the wavelength from 200-700 nm (Fig. 3A). The maximum absorbance peak was found at the wavelength of 259 nm. Moreover, two other peaks were also observed at 280 and 325 nm, which indicates the dispersity of QDs in solution is not the narrow size distribution [29].
As effective mass model has suggested that the absorption band of the
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quantum dots show dependency with radius of particles [30]. Fig. 3B illustrates the
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phosphorescence property of the ZnS QDs in 20mM PBS (pH 8.0). A strong phosphorescence
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peak centered at around 580 nm is observed. It also indicates the monodispersity of QDs in the buffer solution. Lin and his co-worker have also found the emission peak at 590 nm of ZnS QDs
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at room temperature [29]. 3.2.Effect of pH on Mn-doped ZnS QDs
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The phosphorescence intensity of QDs was noted in PBS at different pH ranging from 8 to 11. Table 1 shows that the phosphoresce behavior of ZnS QDs is pH dependent and has enhanced
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with increasing pH. The maximum phosphorescence intensity was found at pH 10. As earlier work also revealed that as pH is elevated the RTP also increases [29]. The chemistry behind this
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is basically deprotonation of carboxyl group because of increase in pH led to doping of Zn(II) occured and create the surface defect that ultimately protects from quencher like dissolve oxygen
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molecules [30]. Second, is the enhancment of coordination between sulfydril and metal ion group with incrase in pH and also lower the process of nonradiative transition, thereby
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enhacement in intensity was observed [27]. 3.3.Optimization of QDs concentration
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As the data shown in Table 2, The best concentration of QDs was found 5000ppm means 10 times diluted from the stock solution of 50,000ppm. Massive concentration of ZnS QDs in sol gel matrices cause agrragation of the particles and the gel becomes cloudy. Due to this formation of bigger nanoparticles are tooke place and resulted loss of phosphorescence intensity [31]. 3.4.Immobilization of enzyme with ZnS QDs Crude enzyme arginine deiminase was co-immobilized with ZnS QDs in the hydrosol gel. SEM analysis of the immobilized disk with enzyme and ZnS QDs was shown in figure 1(C and D). Figure 1C shows that dispersed ZnS QDs in hydrosol gel matrix in regular way and estimated size was found 100 to 500 nm. Different concentrations of standard arginine solution were
ACCEPTED MANUSCRIPT prepared in PBS (pH 8; 0.20 mM). The phosphorescence intensity was measured 545, 580 and 680 nm. As shown in Figure 4, the concentration of arginine decreased the phosphorescence intensity was also decreased due to decrease in pH. A standard chart was prepared using different concentrations of arginine as shown in table 3. Further it is used to estimate arginine concentration of unkown samples. The storage stability of sensing disk was also checked and it is found to be stable around two weeks without significant decreased in performance (table 3).
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3.5.Arginine detection and application in real samples
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The developed biosensor was applied for the detection of arginine with known concentrations of
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arginine 0.01 and 100mM in PBS, and standard chart was prepared (Table 3). The response time of the developed biosensor for arginine detection was found to be 5 min. further, the developed
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biosensor was applied to check arginine in fruit juices samples. The observed result showed that arginine cotents the order of watermelon >guava >pomegranate >orange juice samples. The
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reliability of the system was also checked by using simulated fruit juices and it was observed >90% reliabile. The storage stability of the biosensor was achieved one week, after that there is
4.Conclusion and future perspectives
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decreased activity by 5%.
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The maximum enzyme activity of partially purified enzyme was found to be 42.66±2 IU/ml (specific activity 8.7±0.3 IU/mg). UV-vis spectra of ZnS QDs were observed at 259 to 231 nm.
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Phosphorescence spectra peak of ZnS QDs was observed at 580 nm. Size of the ZnS QDs was found to be in the range of 200 to 1000 nm. ZnS QDs was detected at pH ranges of 8 to 11 and
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phosphorescence intensity was found best at pH 10. The phosphorescence intensity increased with increase in concentration of arginine and pH level confirm the scanning principle. Linear
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range of detection of arginine was found to be 1.0 to 10-4M and developed biosensor was used to monitor arginine in guava, orange, water melon and pomegranate fruit juices. The reliability of developed biosensor was checked with spiked fruit juices samples. The storage stability of device was found to be one week. A novel combination of ZnS QDs with partially purified arginine deiminase modified sensor disk was developed with significant feature for an on the spot biosensor: simplicity, selectivity and low cost in fabrication tgether mak it very usefull approach. Acknowledgment: The authors are grateful to the Department of Biotechnology, Punjabi University Patiala for providing facilities to carry out this work.
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Table 1: Effect of pH on Mn-doped ZnS QDs
pH
Phosphorescence intensity (AU) 655.61 nm
8
539.26
284
8.5
951
9
972
9.5
1306
10
1845
10.5
1647
11
1387
528
532.18 742 957 930.07 760.42
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581.21 nm
Phosphorescence intensity at 580 nm (AU) 1.35 ±0.60 201.43 ±3.85 254.72 ±2.90 28.45 ±4.6
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ZnS QDs concentration for immobilization (ppm) 50,000 10,000 5,000 500 n=3
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Table 2: Phosphorescence intensity of different QDs concentration
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Table 3: Standard chart for arginine with storage stability Arginine concentration (M) 1 10-1 10-2 10-3 10-4 10-5
Phosphoresce intensity at 580nm (AU) 1 week 2nd week 3rd week 235.66 230.11 201.76 210.09 209.38 186.19 187.36 177.10 141.35 156.98 146.45 120.48 124.36 122.95 70.38 112.87 107.71 30.21 st
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Guava Pomegranate Watermelon Orange n=3
180.74 ±2.6 177.28 ±3.1 185.25 ±2.9 168.55 ±4.2
Spiked with 10mM arginine at 580nM 190.86 ±3.6 188.98 ±2.8 197.98 ±3.4 182.30 ±3.7
Concentration of arginine estimated
% reliability
10.16 10.08 10.56 9.72
98.4 99.2 94.6 97.2
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Fruit samples
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Fig.1: SEM analysis of ZnS QDs (A) ZnS QDs in PBS, (B) ZnS QDs in Sol gel, (C) high magnified image of ZnS QDs in Sol gel and (D) immobilized ZnS QDs with Arginine deiminase.
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Fig. 2: ZnS QDs in UV light (A): tube filled with water and (B): aqueous solution of ZnS QDs
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Fig. 3: (A) Absorbance, (B) Emission spectra of Mn-doped ZnS QDs. Solution were prepared in PBS (20mM pH 8.0).
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100 mM
10 micromole
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Intensity (AU)
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0 450
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550 600 Wavelength (nm)
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Fig.4. Phosphorescence response of different concentration of arginine (0.01 – 100mM).