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Summer Research Fellowship Programme of India's Science Academies

Conjugation of surface functionalized gold nano particle with bio molecule to use as a biosensor

Dr. Manash Barthakur

Associate Professor, Department of Zoology, Pub Kamrup College, Baihata Chariali 781381

Professor Parameswar K Iyer

Professor, Department of Chemistry, Indian Institute of Technology, Guwahati 781039

Abstract

Biosensor is a device used to detect the presence and function of biomolecules in different biological fluid. In the field of biosensor development, gold nanoparticle attracts great attention due to unique properties and surface charges. Different biosensors are now in use for clinical application. Development of a biosensor to detect the functional activity of GABA receptor has great demand because of the reason that gabaergic neurons have great contribution in different neurological disorders especially epilepsy, anxiety disorder. Modulation of gabaergic neuronal function can control different neural disorders. The GABA analogue gabapentine is selected as a bio-molecule and conjugated with positively charged gold nanoparticles. Gabapentine has negative zeta potential and hence cationic gold nanoparticles are used for conjugation. Positively charged gold nano particles are prepared from chloroauric acid after reduction with sodium borohydride and CTAB is used as
surfactant. Positively charged gold nanoparticles are attached with gabapentine by electrostatic interaction. The nanoparticles and biomolecules are mixed at the ration of 1:1000. This biomolecule and gold nanoparticle conjugate is attached with a fluorescence dye methylene blue. The formation of gold nanoparticle, gabapentine and methylene blue conjugate was studied by analysis of zeta potential and UV spectra and confirmed by EDX analysis. Gabapentine and gold nanoparticle conjugate can be attached with specific receptor present in gaba neuron and the fluorescence signal released from the methylene blue can help in visualization of the gaba neuron in brain slice under fluorescence or confocal microscope. The conjugate can be used as a basic component for the optical biosensor development to detect the functional activities of the brain in vitro and ex vivo condition.

Keywords: nanoscale matter, interaction, ligand, gabanergic neuron

Abbreviations

Abbreviations
 AuNPGold nanoparticle 
GP Gabapentine 
 CTABCetyltrimethylammonium bromide

INTRODUCTION

Background/Rationale

Identification of bio-molecules in the body fluid or biological tissue is important for proper diagnosis and treatment. Biosensors can often be used to detect the physiological activities of the body. Biosensors have wide applicability in the field of diagnosis of different neural disorder. Although, biosensors are developed for diagnosis of different disorders but the available literature reveals that biosensor for diagnosis of neural disorders are yet to be developed. Antibody based biosensors are already in use (Lee et. al. 2019), while the neurotransmitter based biosensors to detect the structure and function of specific neurons are not available till today. Neurotransmitters are small molecules that are released from the nerve terminals. After release from the nerve terminal neurotransmitters bind with receptor protein and activate the next neuron. Neurotransmitters are later degraded by some enzymes present in the synaptic cleft. There are different types of neurotransmitters some are excitatory and others inhibitory. Inhibitory neurotransmitters are mainly GABA secreted from GABAergic neurons present in the brain. They play an important role in the brain physiology, related to behaviour and psychology of man and animals. Different biosensors are developed recently to quantify the level of neurotransmitter in the brain (Yangguang et. al. 2019). The quantity of neurotransmitter depends upon the number of specific neurons present in the brain. Therefore detection and quantification of location specific neurons are important for diagnosis and treatment of neural disorder and also for new drug development. Present research work has been designed to develop neurotransmitter based biosensor using gold nanoparticles as a linker for identification GABAergic neurons in the brain. The technique is a fluorescence based technique where a fluorescence dye is tagged with the gold nanoparticle and neurotransmitter conjugate.

Statement of the Problems

Study of the activities of GABA neurons is important in research field, because most of the therapeutic preparations are targeted to GABA neuron. It is believed that modulation of functions of receptor of GABA neuron can help in controlling certain neural and neuropsychiatric disorders. The findings of this research work might help the researchers to study the brain activity under different pathophysiological conditions.

Objectives of the Research

Identification of gaba neuron using gaba analogue gold nanoparticle conjugate

Overall objectives

a. Surface functionalized gold nanoparticle preparation.

b. Conjugation of gold nanoparticles with GABAanalogue

c. Tagging of fluorescence dye with gold nanoparticle and gabapentine conjugate.

Review of Literature

Application of nanoparticles for diagnosis and treatment of disease is an emerging field of research and scientists are trying to developed different technique for treatment of cancer. In biological system, different toxic metabolites are formed due to different enzymatic reactions in the body. Hence, nanoparticle based detection techniques for different by-products of enzymatic reactions have been developed. Positively charged gold nanoparticles are prepared and used for detection of hydrogen peroxide in the body (Khan & Cho, 2018). Gold nanoparticles can be stabilized by albumin, and the albumin stabilized gold nanoparticles can be conjugated with antibodies. The antibody conjugated gold nanoparticles can be applied for detection of bioavailability of different bio-molecules (Nghiem et.al. 2010). Surface functionalized gold nanoparticles for detection of different biomolecules in the human blood are investigated (Faham et. al. 2017). Scientists are trying to synthesize less toxic positively charged gold nanoparticles for delivery of different drugs to the specific parts of the body. Ghost RBC and nanoparticle conjugation is also possible (Gau, et. al. 2013). Use of gabapentine in the field of neuro-therapy is highly practiced (Dhana Lakshm et. al. 2012). However, the application of gabapentine for sensor development is a new concept. Gold nanoparticles can be used for colorimetric assay because colour of the gold nanoparticle depends on the particle size. Therefore different attempts are made to use gold nanoparticle based sensor for colorimetric estimation (Chang, et. al. 2019). Biological recognition probe is developed after conjugation of peptide with gold nanoparticle and the probe can be used for detection of biological component after interaction and formation of nanoparticle aggregation. Aggregated nanoparticles can be detected by colorimetric method due to blue shifting. Versatile surface chemistry of gold nanoparticle provides a significant help to link with different molecular probe having thiol group for conjugation and detection of biological membrane. Gold nanoparticle based colorimetric biosensors are mainly aggregation based colorimatric detection, etching based colorimetric detection, nanoenzyme-based colorimetric sensing and growth-based colorimetric sensor (Chia-Chen Chang et. al. 2019).

Organic electronic multifunctional devices are developed to execute chemical stimulation and electrical sensing. Organic microelectrode are prepared to control different neural firing by delivering GABA to the targeted sites (Jonsson et.al. 2016). Identification of normal and polymorphic GABA receptor is very important because the polymorphism in GABA receptors has undesired impact towards the development of different neural disorder (Yang. et. al., 2017).

Methods: Synthesis of positively charged gold nanoparticle was done following some modification of earlier methode (Faham et. al. 2017)

Preparation of CTAB capped gold nano particles.

a) Cetyltrimethylammonium bromide (CTAB) is a phase transfer molecule functions as detergent for stabilizing the salt in organic phase. Electrostatic layer by layer assembly involving opositly charged surfactant can nanoparticle suitably functionalize surfaces.

b) Preparation of CTAB stabilized gold nanoparticle synthesis: A mixture of 0.25 ml of 0.01 M HAuCl4, 9.75 ml of 0.6 M, CTAB was kept under continuous stiring for 1 hour. Gold chloride solution of 0.01 M was prepared and kept in 2 degree centigrade. 250 μl of gold chloride solution was added in to the CTAB solution and the mixture was under continuous stirring. After two hours of continuous stirring, 120 μl of 0.1 molar ice cold sodium borohydride was added drop by drop. The colour of the solution changed from yellow to wine red. The stirring is continued for 24 hours.

c) Preparation of Gabapentine solution: Gabapentin (molecular weight 171.237gm/mol)stock solution 29.19 mM was prepared by dissolving 250 mg of gabapentine in 50 ml of milliQ water. the solution was kept for future use.

gabapentine.jpg
    Schematic representation of the base molecule. 

    d) Preparation of methylene blue stock solution: Commercially available 0.1% methylene blue solution was used as stock. Working solution of methylene blue was prepared by dilution of stock methylene blue with distilled water and the solution was made at 150 μM.

    Conjugation of CTAB capped gold nanoparticles with gabapentine: For conjugation of gabapentine with gold nanoparticles, gold nanoparticles and gabapentine are mixed at 1:1000 ratio in a beaker on a magnetic stirrer and the stirring was continued for 24 hours. The methylene blue solution was mixed with the solution at the ratio of gold and methylene blue 1:1000. The mixture was continuously stirring for 24 hours and the mixture was centrifuged at 12,000 rpm for 1.30 hours. The supernatant containg unbound methylene blue and gabapentine was discartded and the precipitate was dissolved with miliQ water. The conjugate was stored at room temperature for future application.

    AuNP paint final 1.jpg
      CTAB capped gold nanoparticle conjugation with gabapentine

      RESULTS AND DISCUSSION

      Spectroscopic Study

      Methylene blue, CTAB coated gold nanoparticle, gabapentine and conjugate were studied in the UV-Vis spectrophotometer. CTAB coated gold nanoparticle showed peak absorption at 526 nm. The λ max of gabapentine was found 260 nm. The methylene blue showed λ max 662. After conjugation with gabapentine the λ max of the conjugate shifted and peak absorption found at 558nm. The peak absorption of the conjugate tagged with methylene blue showed 533.

      mb_1.JPG
        UV- spectra of the methyline blue.
        All.JPG
          UV-Vis spectra of gold nanoparticle, gabapentine, Gabapentine-goldnanoparticle conjugate and gabapentine-goldnanoparticle conjugate attached with methyline blue.

          Photoluminscence Study

          Photoluminiscence characteristics of the CTAB stabilized goldnanoparticles, methylene blue, gabapentine, methylene blue gold nanoparticle conjugate and gold nanoparticle gabapentine and methylene blue conjugates was studied. Methylene blue showed strong fluorescence activity at the excitation of 640 nm and emission range from 640 nm to 900nm (Fig. 5). Fluorescence characteristics was not found in the conjugate of gold nanoparticle gabapentine and methylene blue (Fig. 9). Gabapentine and methylene blue conjugate showed fluorescence activity under excitation of 650 and emission 650 to 900 (Fig. 7).

          PL-MB.JPG
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          PL spectra of methyline blue
          Slide2_3.JPG
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          PL spectra of ctab capped gold nanoparticle. Nanoparticles were excited at 360 nanometer and the emission recorded from 360 to 600 nm range. 
          Slide3_3.JPG
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          PL-spectra of gold nanoparticle and methylene blue conjugate. The conjugation was excited at 650 nm and emission recorded from 650 nm to 900 nm. The excitation wave length was selected on the same wavelength with the methylene blue. 
          Slide4_2.JPG
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          PL spectra of ctab capped gold nanoparticle and gabapentine conjugate. The conjugate was excited at 360 nm wave length and emission range was recorded upto 600 nm.
          Slide5.JPG
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          PL spectra of gold nanoparticle, gabapentine and methylene blue conjugate. The conjugate was excited 360 nm and the emission range was recorded from 360 to 600 nm. 

          Zeta Potential Analysis

          Surface charge of CTAB stabilized gold nanoparticles were studied in Zeta analyser and was found that the ctab capped gold nanoparticles had positive charface charge and the average value was +35.2 mv. The gabapentine molecules are negatively charged and the average value of zeta potential of Gabapentine was found -17.2 mv. After conjugation with the positively charged gold nanoparticles the surface charge of the cojugate was found 0.171 (mV).

          GNP-CTAB P.jpg
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          Zeta potential of CTAB coated gold nanoparticles. .
          GP-ZETA.jpg
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          Zeta potential of Gabapentine. Gabapentine shows negative zeta potential.
          Slide2_2.JPG
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          Zeta potential value of gold nanoparticle and gabapentine conjugate. The peak zeta value shows slightly positive.
          AuNP-MB.jpg
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          Zetapotential value of methylene blue and gold nanoparticle conjugate.
          Slide4_1.JPG
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          Zetapotential value of the gold nanoparticles, gabapentine, and methylene blue conjugate. The peak value of the zeta is slightly negative.

          Stability Study

          Stability of the surface functionalized gold nanoparticle were studied for three days. UV spectra were recorded regularly for three days and λ max was measured and found almost same value with the day 1. The stibility of the gabapentine and gold nanoparticle conjugate was studied using UV-Vis Spectrophotometer. The conjugate was found in stable condition for longer duration but after conjugation with methylene blue the conjugate lost the stability after 2 days and found as precipitate.

          EDX Analysis

          Energy dispersive microanalysis of Gold nanoparticles, gabapentine and methylene blue conjugate was studied. The conjugate showed the different peaks for sulphur and carbon. Presence of sulphur and carbon peak confirm the formation of conjugate.

          r1.JPG
            Scanning Electron Microscopic structure of the conjugate
            r2_2.JPG
              Signal received from different element of the conjugate.

              DISCUSSION

              The CTAB is a phase transfer surfactant and a detergent can be used for synthesis of surface functionalized gold nanoparticles. Positively charged gold nanoparticles are synthesized from gold chloride reduced by sodium borohydride and oppositely charged surfactant assembled layer by layer can functionalize surfaces of the nanoparticles. Previously CTAB is used for gold nanoparticle seed preparation and also used for positively charged gold nano-cluster preparation (Faham et. al. 2017). The surface charge of nanoparticles prepared in this experiment shows positive as observed from the zeta potential value. The CTAB functionalized gold nanoparticles can bind with gabapentine through electrostatic interaction as the zeta potential of the gabapentine is strongly negative and the ionic bonds might be formed among these two molecules. The Gabapentine and CTAB functionalized gold nanoparticle conjugate is almost neutral in surface charge characteristic as observed in zeta potential value. The conjugate of gold nanoparticles and gabapentine shows fluorescence characteristic might be due to sharing of lone pair electron or the aggregation of gold nanoparticle after conjugation with gabapentine. The conjugate is stable for longer duration indicating that the conjugate can be applied as biosensor or pharmaceutical device. The conjugate tagged with strong fluorescence dye-methylene blue and the conjugate characteristics were studied. After tag with methyline blue the tag conjugate losses fluorescence characteristics as observed in PL spectra. This might be due to the ionic imbalance and participation of lone pair electron in bond formation as because the conjugate losses the stability. Surface charge of the nanoparticle is important for interaction of biomolecules with nanoparticles. It has been reported that positively charged nanoparticles absorb protein with isoelectric point <5.5 while negative surface charge increases the protein absorption with isoelectrict point > 5.5. Moreover, the attachment of biomolecules to the surface of nanoparticles also depends upon surface area to volume ratio (Phogat et. al. 2018). It is also reported that after conjugation with charged nanoparticles, denaturation of protein takes place, while in presence of neutral surface, protein retains the original characteristics. Besides, surface morphology also plays an important role in nanoparticle biomolcular
              conjugation. Absorption of protein on the surface of the gold nanoparticle causes reduction of surface charge (Liu & Peng, 2017). Changes of the surface charge of gold nanoparticles after conjugation with gabapentine was also observed in this experiment.

              CONCLUSION

              This research work carried out contributes knowledge towards the conjugation of negatively charged biomolecules with surface functionalized nanoparticles. But the applicability of the fluorescence dye conjugated bio-molecules in animal model is yet to be ascertained. Identification of receptor at molecular level cannot be done with fluorescence dye conjugate. Future works at molecular level would contribute more information in the applicability of the conjugate. The neurotransmitter based nanoparticle conjugate might have some pharmacological importance to control neural disorders. The findings of the present research work would provide valuable clue for development of biosensor for detection of excitable and non-excitable cells. Since the nanoparticles can cross the blood brain barrier, hence the conjugate might be used to study in vivo experiment.

              Future Scope

              1. We can study the conjugation of different biomolecule and nanoparticles with different surface charges and their application in diagnosis and as therapy.

              2. Further magnetic nanoparticles can be used to conjugate with different biomolecules and has the possibility to develop a device for separation of biomolecules from tissue extract.

              3. There is possibility for use of receptor specific biomolecules and nanoparticle conjugate in cancer therapy.

              4. It is to be find out whether receptor specific biomolecule and nanoparticle conjugate would be applied for in vitro, in vivo and ex vivo experiment.

              REFERENCES

              1. Lee, JH., Chae, EJ., Park, S. et al. Nano Convergence (2019) 6: 13. https://doi.org/10.1186/s40580-019-0184-3

              2. Yangguang Ou, Anna Marie Buchanan, Colby E. Witt and Parastoo Hashemi Frontiers in electrochemical sensors for neurotransmitter detection: towards measuring neurotransmitters as chemical diagnostics for brain disorders: Anal. Methods, 2019, 11, 2738–2755

              3. Mohammad Mansoob Khan & Moo Hwan Cho (2018): Positively Charged Gold Nanoparticles for Hydrogen Peroxide Detection. BioNanoScience (2018) 8:537–543

              4. Thi Ha Lien Nghiem, Thi Huyen La, Xuan Hoa Vu, Viet Ha Chu, Thanh Hai Nguyen, Quang Huan Le, Emmanuel Fort, Quang Hoa Do and Hong Nhung Tran (2010) Synthesis, capping and binding of colloidal gold nanoparticles to proteins: Adv. Nat. Sci.: Nanosci. Nanotechnol. 1 (2010) 025009 (5pp) doi:10.1088/2043-6254/1/2/025009

              5. R. Faham, A. Samadi, and J. Abolhasani ( CTAB-Capped gold nanoparticles as a new probe for spectrophotomatric determination of heparin, 2017. Journal of Applied Spectroscopy, Vol. 84, No. 3, July, 2017.

              6. Jianfeng Guo, Mark J Armstrong, Caitriona M O’Driscoll, Justin D. Holmes and Kamil Rahme ( 2015). Positively charged Surfactant free gold nanoparticles for nucleic acid delivery: RSC, Adv. ,5, 17862 –17871.

              7. Dr. Weiwei Gao, Dr. Che-Ming J. Hu, Ronnie H. Fang, Brian T. Luk, Dr. Jing Su, and Prof. Liangfang ZhangSurface Functionalization of Gold Nanoparticles with Red Blood Cell Membranes. Adv Mater. 2013 12; 25(26): 3549–3553. doi:10.1002/adma.20130063

              8. Dhana Lakshmi. P, Rahul Nair, Chakrapani. M, Venkatkrishnakiran. P (2012) Solid of lipid nanoparticle system for delivery of drugs to the brain. International Journal of Biopharmaceutics. 2012; 3(2): 70-77.

              9. Chia-Chen Chang, Chie-Pein Chen, Tzu-Heng Wu, Ching-Hsu Yang, Chii-Wann Lin and Chen-Yu Chen (2019) Gold Nanoparticle-Based Colorimetric Strategies for Chemical and Biological Sensing Applications: Nanomaterials 2019, 9, 861; doi:10.3390/nano9060861

              10. Amanda Jonsson, Sahika Inal, Ilke Uguz, Adam J. Williamson, Loïg Kergoat, Jonathan Rivnay, Dion Khodagholy, Magnus Berggren, Christophe Bernard, George G. Malliaras, and Daniel T. Simon,(2016) Bioelectronic neural pixel: Chemical stimulation and electrical sensing at the same site: PNAS 23, (113). 39440–9445

              11. Shuhan Yang, Xuan Guo, Xiaopeng Dong, Yu Han, Lei Gao, Yuanyuan Su, Wei Dai, and Xin Zhang (2017) GABAA receptor subunit gene polymorphisms predict symptom-based and developmental deficits in Chinese Han children and adolescents with autistic spectrum disorders. Sci Rep; 7: 3290 doi: 10.1038/s41598-017-03666-0.

              12. Faham R. Faham, A. Samadi, and J. Abolhasani (2017), CTAB-Capped gold nanoparticle as a new probe for spectrophotomatric determination of heparin. Journal of Applied Spectroscopy, 84, 3, 425-426.

              13. Navneet Phogat, Matthias Kohl, Imran Uddin and Afroz Jahan ( 2018) Interaction of Nanoparticles with Biomolecules, Protein, Enzymes, and Its Applications: Precision Medicine Tools and Quantative Approaches : 253-276

              14. Liu, J., Peng, Q., Protein-gold nanoparticle interactions and their possible impact on biomedical applications, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.03.055.

              ACKNOWLEDGEMENT

              I profusely thank Prof. Parameswar K Iyer, Department of Chemistry, IITG, for giving me the opportunity to work in his laboratory to complete my research project. His invaluable technical support and encouragement are key factors that have made my research programme
              a successful one. I shall remain grateful to Mr. Subrata Mondal and all the lab members for continuous support and healthy discussion during my experimental study period. I also express my thankfulness with sincerity to IASC-NSAI-INSA for offering me the fellowship
              and continuous support throughout the programme. I also express heartfelt gratitude and thanks to the authority of Indian Institute of Technology, Guwahati for allowing me to do the research work utilizing their laboratory facilities including Central Instrumentation Facilities.

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