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The main investigator and author of this project is an Erasmus Mundus Master's student at University of Lincoln, leading in a consortium of two other universities including University of Cordoba and Institute of EGAS MONIZ. Previously, author holds a 3-year BSc degree in Chemistry and 1-Year MSc in Analytical Chemistry from University of Karachi, Pakistan. His research interests include development of novel nanomaterial assemblies and their applications in analytical and bio-analytical sciences, aiming to develop µ-TAS or LAB-on-Chip.
The project is being supervised by Prof. Dr. Juan Manuel Fernández-Romero, a professor at Campus de Rabanales, University of Cordoba, Spain. Key features of his current research include automated analytical methods: fast-kinetic methods, continuous flow analysis and micro and nanofluidic devices and chromatographic and non-chromatographic techniques using pre- on- and post- column derivatization coupled with luminescence detection.
Table of Contents
Apart from biological importance, reduced organic sulfur (ROS) or thiols (R-SH), mainly Glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), play an important role in our ecosystem. Their affinity towards toxic heavy metals like mercury can be used for its detoxification. Harris et al. has already confirmed the presence MeHg-RSH complexes in different aquatic samples (Harris, Pickering, & George, 2003). In past years, environmental thiols analysis was hindered due to rapid oxidation of these sulfhydryl compounds to disulfides. The resulting compounds are no more target for labeling or bio-conjugation and their chemisorption kinetics with gold is much reduced (Bain, Evall, & George M. Whitesides, 1989). Moreover, low concentration levels (nM or sub-nM) of thiols in aquatic environment and complex matrix interferences have further hampered the task. Different attempts have been made to solve these issues by using highly sensitive state of the art instrumental technique along with an additional preconcetration step (Zhang, Wang, Rt, House, & Page, 2004), (Tang, Wen, & Santschi, 2000). Florescent (Tang, Shafer, Vang, Karner, & Armstrong, 2003) and electrochemical (Al-Farawati & Van den Berg, 2001) methods were also employed to increase the sensitivity and limit of detection.
However, recent developments in the field of nanoscience and microfluidics led us to highly selective, sensitive and portable devices that can be effectively used for on-spot environmental monitoring (Jokerst, Emory, & Henry, 2012) consequently solving the issue of rapid oxidation. By reducing sample and solvent volumes, these microfluidic assemblies can reduce the time of laborious analytical processes (i.e separation, isolation, chemical and biological reactions) to mere few minutes or even to seconds making them efficient, cost effective and less toxic (Lafleur, Senkbeil, Jensen, & Kutter, 2012). Rapid growth in this field can also be witnessed by the fact that various international journals have dedicated themselves for publishing microfluidic research, mainly related to the fabrication of new platforms. Main goal of the researchers is to develop a µ-TAS or Lab on a chip bearing in mind their applications in field of science.
Beside, development of new hybrid nanomaterial with higher stability, tunable size, physiochemical properties and easy separation (in case of MNPs) have extended their applications from its origin in analytical chemistry (as sensitive and selective detection probes) to chemical syntheses, biochemistry, and medical sciences (J. Chen et al., 2007), (S. Chen, Chang, & Road, 2004), (Guo et al., 2008), (Saha, Agasti, Kim, Li, & Rotello, 2012), (Yang et al., 2012), (Liang et al., 2011).
Saha et al. (Saha et al., 2012) has recently highlighted the importance of gold nanoparticles in the field of chemical and biological sensors by discussing some of the physical properties of NPs that can be used to develop various sensing strategies. Among them is a fluorescence quenching property of gold nanoparticles which is now widely used as a detection strategy in bio-analytical applications (Yang et al., 2012), (Lafleur et al., 2012)(S. Chen et al., 2004). On the other hand, Sharma et al. and Akbarzadeh et al. have reviewed some of the recent advancements in magnetic nanoparticles and their applications in biomedical and electronic industries (Akbarzadeh, Samiei, & Davaran, 2012), (Sharma, 2011). Authors highlighted the advantage of using easy to separate magnetic nanoparticles like Fe3O4 and Co3O4 nanospheres but later criticized their use in biomedical applications due to their toxicity. Considerable efforts have been made in functionalizing nanoparticles for obtaining higher stability in physiological environment, reducing their toxicity and recognition of biochemical species. Thanh et al. (Thanh & Green, 2010) have rationalized the importance of biofunctionalisation around inorganic metal, semiconductor and magnetic nanoparticles discussing variety of polymer coatings that can be used in different type of core spheres. Besides, different fluorescent polymers, dyes and resins can also be coated in order to obtain luminescent MNPs that can be used as a detection probe in different fields of analytical sciences (S. Chen et al., 2004).
Due to the availability of wide range of nanoparticle with their size-dependent colorimetric, magnetic and fluorescent properties, integration of two or more nanoparticles (fluorescent and magnetic) on a micro level scale can be used as a detection probe on a microfluidic platform for environmental monitoring (Selvan, Patra, Ang, & Ying, 2007). In spite of various researches carried out on microfluidic determination of environmental contaminants (Jokerst et al., 2012)or magnetic hybrid gold nanoparticles based determination of biological analyte (Table 1), there is no such study that uses integrated magnetic functionalized gold nanoparticles as a detection system on microfluidic device for the determination of thiols in environment samples. Hence, we are proposing an On-chip determination method for the analysis of Glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), using Fe3O4 NPs coated with a fluorescing PFR shell and loaded with AuNPs (as quenching material) making a magnetically separable fluorescing probe with low toxicity.
Table 1: Microfluidic analysis performed for real environmental samples (Li & Lin, 2009)
Pulse amperomertic detection
Aims and objectives
The central aim of the project is to synthesize a hybrid nanostructure by successive coating of phenol-formaldehyde (PFR) resin, PEI/PSS/PEI polymer and gold nanoparticles on Fe3O4 magnetic nanospheres. As-synthesized nanostructure thus will be used as a Turn-on detection probe on standard microfluidic device for the determination of forensically important environmental thiols.
The outcomes or objectives of this research include
Development of a simple proto-type hybrid-MNPs based microfluidic assembly for sensing environmental thiols Glutathione (GSH), homocysteine (Hcy), and cysteine (Cys) in aquatic samples.
Optimization and validation of these magnetic fluorescing nanoprobes based micro device as a sensitive, selective and robust detection system for the determination of forensically important environmental thiols.
Outlined project includes synthesis of multifunctional magnetic hybrid Fe3O4 nanospheres loaded with gold nanoparticle and their use as a detection probe on a standard microfluidic device operated by syringe driven system (SDS). The multifunctional nanostructured fluorescent probe consist of ferric oxide (Fe3O4) magnetic core, a green-luminescent phenol-formaldehyde resin (PFR) shell with an extra coating of polyethylene-imine (PEI)/poly (sodium 4-styrenesulfonate) (PSS)/polyethylene-imine (PEI) and an outmost coating shell composed of AuNPs. Purpose of an extra coating of PEI/PSS/PEI is to alter the negative charge of [email protected] complex into positively charged [email protected]@polymer consequently creating an electrostatic attraction between [email protected]@polymer and negatively charged AuNPs. Detection is based on phenomena of fluorescence resonance energy transfer (FRET) in which strong association of thiol with AuNPs separate [email protected]@polymer from AuNPs and recovered the fluorescence of [email protected]@polymer which was initially quenched due to electrostatic attraction between complex and AuNPs (Figure 2). The quenching efficiency of AuNPs can be calculated using Equation 1
Î q = F - F0 / F0
While recovered efficiency can be calculated using Equation 2
Î r = (Fa - F) / (F0 - F)
F = Fluorescence intensity of [email protected]@polymer without AuNPs
F0 = Fluorescence intensity of [email protected]@polymer with AuNPs
Fa = Recovered fluorescence intensity
Finally, as-synthesized probe will be tested on standard two channel microchip attached to pumping assemble. Optimization and validation of the detection system will be conducted using different parameters discussed in table 2.
Figure 2: Scheme of the project showing the detail methodology to be followed in synthesizing nanoprobes. PAA = (Poly-acrylic acid); BSA = (Bovine Serum albumin; PFR = (phenol-formaldehyde resin); R6G = (Rahidamine 6G). Magnetic nanoparticles will be prepared in aqueous medium. (For detailed explanation see 3.1).
The project has been divided into three major parts (Figure 1) including
Synthesis of magnetic hybrid fluorescing probes
Characterization of as-synthesized nanostructures
Optimization and validation of the proto-type microchip.
Figure 1: Classification of the project
Synthesis of Hybrid magnetic fluorescing probes
The synthesis of these hybrid nanostructures require various important steps including preparation of seed (metallic core sphere), preparation of gold nanoparticles, coating of phenol-formaldehyde (PFR) resin, PEI/PSS/PEI polymer and final loading of gold nanoparticles on the surface of fluorescing complex.
Selections and preparation of Fe3O4 magnetic core sphere
The toxicity and biocompatibility of nanomaterial (i.e mainly MNPs) lies in the nature of magnetically responsive metal like iron, manganese, zinc, nickel, and cobalt. In spite of having high magnetic properties, cobalt, manganese, zinc and nickel are of little interest in biomedical and environmental applications due to their toxicity and oxidative susceptibility. However, Iron oxide nanoparticles are most commonly used as a stable magnetic seed due to their satisfactory magnetic response and low toxcicty.
The magnetite Fe3O4 clusters can easily be prepared using solvothermal method by dissolving FeCl3â‹…6â€‰H2O in an appropriate volume of ethylene glycol (50-100 mL) followed by the addition of sodium acetate or citrate and polyethylene glycol. The solution is stirred vigorously for 30 mins and autoclaved at 200â€‰°C for 8-72 h. Final product is washed several time with ethanol and dried at 60â€‰°C for 6 h (Deng et al., 2005).
Note: The amounts and volume of solvents should be added quantitatively as they affect the size of nanostructures. The whole process can be accelerated using microwave(You et al., 2012).
Selection and preparation of gold nanoparticles:
Among other noble metal, AuNPs have some distinct physical and chemical properties including high stability, unique optoelectronic properties, high surface-to-volume ratio and excellent biocompatibility using appropriate ligand. These properties can be varied by changing the surrounding of AuNPs which makes it excellent scaffold as a detection probe or a part of detection system (Saha et al., 2012).
Gold nanoparticles, as quenching element, can be prepared by reducing 1 ml of 1.0 wt % HAuCl4â‹…3H2O with 0.1% NaBH4 solution, prepared by dissolving NaBH4 into 1.0 wt % sodium citrate solution, in 100 ml of deionized water and 2 ml of 1.0 wt % sodium citrate solution. The solution is stirred for some while after each addition and finally allowed to stand for 10 mins in vigorous stirring.
Note: Both nanostructures (i.e Fe3O4 and Au) should be made in aqueous solution which allow easy bio-functionalization of these NPs (Thanh & Green, 2010).
Functionalization of magnetic nanoparticles
Different kinds of polymers have been used for functionalizing magnetic nanoparticles to enhance the stability and fluorescence. Thanh et al. has detailed some of the important polymers used as functionalizing ligand of magnetic nanoparticles. Among all, phenol-formaldehyde have some advantage due to it easy coating with different functional nanoparticles and nontoxicity. Furthermore, its high fluorescence makes it attractive nanoprobes for detection and optical targeting.
Coating can be performed by quantitative addition of MNPs, phenol, hexamethylenetetramine (HMT) and poly sodium styrene sulfonate (PSS) in appropriate amount of water. After stirring, solution can be transferred to Teflon-lined autoclave and heated to 160 0C for 6 h. The solution can be purified with ethanol and deionized water.
Modification of [email protected] NPs
As-synthesized [email protected] nanostructure has a negative surface charge density and cannot be decorated with negatively charged AuNPs. Hence, the surface of nanocomposite was modified with consecutive coating of PEI/PSS/PEI. To do so, particular amount of PFR-NPS can be mixed with PEI stock solution, prepared in NaCl solution, vortex, and kept in a dark place for 30 min. Later excess polymer can be can be removed by centrifugation and polymer coating PFR nanocomposites can be re-dispersed in 1 mL of DI water for further studies. Same procedure can be repeated for coating of PSS and finally PEI. Finally, nanocomposites were further modified by mixing AuNPs synthesized in section â€¦â€¦, in an appropriate volume of micro-centrifuge tube, to obtain magnetic fluorescent hybrid nanoprobes. The adherence of AuNPs on the surface of [email protected]@polymer is due to electrostatic attraction between two nanostructures.
Characterization of nanoprobes
Different ways of characterizing nanoprobes, during the process of synthesis, are possible which includes transmission electron microscopy (TEM), X-ray fluorescence spectroscopy (XRF), scanning electron microscopy (SEM), fourier transformation infra-red spectroscopy, X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy. The purpose of characterization is to verify shape and sizes of nanoprobes elements (seeds, resin, and polymer) as they effect the fluorescence intensity of the nanoprobes consequently effecting sensitivity. Each technique provides unique information that can aid in characterizing nanostructures. In our proposed study, we shall use transmission electron microscopy (TEM) with CM-10 Philips Microscope, 0.5nmÃ-0.34nm resolution and equipped with a digital mega-view III camera. The XPS measurements nanocomposites shall be carried out using a Specs Phoibos 150 MCD instrument equipped with a monochromatized AlK (12KV) while morphology of the nanoparticles will be studied using scanning electron microscopie (SEM) (Jeol JSM 6300) microscope operated at 20 KV. Finally, EDAX measurements shall be made to determine the elemental composition.
Note: All instruments are available at central service laboratories of Rabanales Campus, University of Cordoba. Other instrumentations can be used if needed
Application of nanoprobes on a microfluidic chip
Various designs of a microfluidic systems, ranging from simplest model to complex systems, have been published as detection platform including those that are presented by Lafleur et al. (Lafleur et al., 2012) and Li et al.(Li & Lin, 2009). Figure 3 describes our design of microfluidic assembly which includes micro-reactor model FC_R150.332.2 with dimensions of 12Ã-24 mm and an internal volume of 6 Î¼l assembled to a fluidic chip holder (4515) (Micronit, The Netherlands, www.micronit.com), KDS220 syringe pump for controlling flow of the reagents (KD Scientific Inc., MA, USA, www.kdscientific.com), and an optic fiber bundler assembled to a Cary Eclipse Varian spectrofluorimeter (Walnut Creek, CA, USA), as a CL detector. The optic fiber bundler and the microchip would be adapted to an X-Y-Z positioner (Oriel Instruments, USA, www.newport.com/oriel/) to allow fine positioning adjustments.
The working principal of the microfluidic assembly is based on propelling reagent solutions at an optimized flow rate using syringe driven system (SDS) through an injection valve (IV1) while second injection valve (IV2) provides a cleaning cycle between each determination. After pumping required reagents in micro-reactor chip, where reaction of thiols and nanocomposites take place, signal is recorded at optimized wavelength with an optic fiber (OF) connected to the luminescence detector. The OF is adapted to a micrometrical system which performs translational movements in a 3-D space extended along the X-Y-Z axes.
Figure 3: Design of microfluidic assembly. S = Sample syringe; WS = Washing syringe; NPs = Nanoparticles syringe; SDS = Syringe driven system; W-1 = Valve 1; W-2 = Valve 2; OF = Optical fiber attached to XYZ positioner; CLD = Chemiluminescence detection system; PC = Personal computer
Method Validation and optimization
Different validation parameters can be assessed to check the validity and fitness of the fluorescing nanoprobes microchip by analyzing glutathione (GSH), homocysteine (Hcy), and cysteine (Cys) standards, marine and fresh water samples. The analytical signal can be a difference or a ratio (Fa / F0) in fluorescence intensity of nanocomposite before and after the addition of thiols. Analytical signals shall be averaged for at least three measurements.
Variables affecting synthesis of MNPs
Different variables affecting the size and shape of the as-synthesized nanoparticles have been reported. Yang et al., You et al., and Guo et al. have investigated some of the important factors which affect the structure of nanocomposites (Yang et al., 2012)(You et al., 2012)(Guo et al., 2008). We are proposing to optimize the amount of phenol and formaldehyde using response surface diagram (See Section 7.9, (Miller & Miller, 2010) and shall use Two-factor ANOVA to investigate any interaction between these variables. Effect of temperature, reaction time and pH will be optimized using univariant method.
Variables affecting analysis of thiols using microfluidic assembly
All variables regarding spectrofluorimeter detection system, microfluidic assembly and pumping system will be optimized using univariant method of analysis. Ranges of different parameters along with their reasons of selection that can affect thiol analysis are discussed in table 2. Moreover, method shall be validated using different validation parameters like LOD, LOQ, precision, selectivity, calibration and recovery.
For LOD and LOQ, 10 blanks samples in 1-5 µL range can be pumped using sample syringe and allowed to react in microchip. Each sample can be detected in triplicate and averaged. The formula of calculating LOQ and LOQ is mentioned in table 2. Inter-day and Intraday analysis shall be performed to evaluate precision of the method using three different levels of standard concentrations. Nested experimental design shall be used by analyzing six samples in same day as well as in different days. Final results will be expressed in % RSD. Calibration curve shall be plotted between thiol concentration and the difference or a ratio (Fa / F0) in fluorescence intensity of nanocomposite before and after the addition of thiols. Six standard levels in the range, depending on LOQ and LOQ, will be used with each standard level analyzed in triplicate to perform regression analysis. Recovery studies shall be conducted; using pre-analyzed sample, by spiking extra 25, 50, and 75% of standard thiols and analyzing by propose method. Finally, selectivity of the method will be checked by injection different solution of copper, mercury and Lead to observe any significant difference between the intensity of fluorescing probe. Student t-test shall be performed to evaluate the difference between observations obtained from suspected interfering metals.
Table 2: Optimizing parameters involved in the analysis of thiols
Proposed range to be studied
Reason of selecting given ranges
Amount of formaldehyde
(You et al., 2012)
Amount of Phenol
0.015- 0.5 mL
(You et al., 2012)
Effect of temperature
(You et al., 2012)
Effect of reaction time
(You et al., 2012)
Emission spectra lies in this range
To obtain higher signal
Using computer software
Emission slit length
To obtain higher signal
Using computer software
To get maximum fluorescence intensity
Using computer software
of the optic fiber in the microfluidic system
Appropriate displacement in all dimensions
Using computer software
0.1-2.0 Î¼L sâˆ’1
To provide enough time in micro-reactor for reaction
SDS system (Univarient method)
Total volume of the channel is 6 Î¼L
SDS volume (Univariant method)
Effect of concentrations of NPs
1-500 Î¼mol Lâˆ’1
Concentrated enough to detect thiols
Effect of pH
(Sierra-Rodero, Fernández-Romero, & Gómez-Hens, 2012)
Analytical validation parameters
Limit of detection
0.1-10 Î¼mol Lâˆ’1
Six injections of blank sample and using the formula, LOD = blank + 3.3 SD; LOQ = 3 LOD
Limit of quantification
Depends on LOD
Less than 2.5% (ICH)
Most conservative range
Repeatability, intermediate precision and reproducibility (n=6)
Copper, Mercury, lead
Mostly present in river water
Adding interfering species in sample and injecting in microfluidic chip.
Depends on LOD
Regression curve with six standard levels each in triplicate
Three concentration levels (Initial, middle and end levels of calibration graph)
The project is funded by Education, Audiovisual and Culture Executive Agency (EACEA) of the European commission under the supervision of Dr Jose Gonzalez-Rodriguez, Associate professor of the University of Lincoln, as coordinator and program leader of Consortium of the Masters in Forensic Science 512007-1-2010-1-UK-ERA MUNDUS-EMMC of the European Commission under the frame work partnership agreement 2011-0170.
Research benefit to the society
Determination of chemical and biological environmental contaminants like thiols plays a vital role in the field of forensic and environmental sciences, consequently generating a need of a sensitive and selective detection system. Integration of these functionalized nanomaterials (as detection probes) with microchip technology provides us cutting edge detection systems. Moreover, these advance systems offer quick and cost effective analysis in the field of bio-medical, environmental and forensic sciences. Future research in this area may lead us to the development of microchips that can be used by individuals as a selective detection tool in their daily life.
Time management Scheme
Below is the Ghent Chart showing distribution of time regarding various task involved in the project
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