PREPARATION OF BIOCONJUGATED GOLD

NANOPARTICLES USING S1 SUBUNIT PROTEIN FROM SARS

COV-2 VIRUS FOR POTENTIAL DIAGNOSTIC
APPLICATIONS

Al Ameen Sikkandar
M. Tech. Nanoscience and Technology
Centre For Nanoscience and Technology

Under the Guidance of

Mr. R. Selvaraj, Teaching Fellow, CNST, Anna University, Chennai
and
Dr. Prince. R. Prabhu, Ph.D., Scientist, Universität Hamburg, Germany
1
 INTRODUCTION

• Gold colloid is a suspension consisting of sub-micron gold nanoparticles suspended within a solvent, most often water. Gold
nanoparticles have unique optical, electronic, and thermal properties and are being incorporated in a wide variety of technologies
including microscopy, electronics, diagnostics, and therapeutics.

• Gold nanoparticles (AuNPs) have been widely employed in the field of bionanotechnology based on their unique properties and
multiple surface functionalities. The ease of AuNP functionalization provides a versatile platform for nanobiological assemblies with
oligonucleotides, antibodies, and proteins.  

• Bioconjugates of AuNPs have also become promising candidates in the design of novel biomaterials for the investigation of biological
systems.

• The versatility of AuNPs has provided useful materials for a range of biomedical applications. In diagnostics, the binding event

between the analytes and the AuNPs can alter the physicochemical properties of AuNPs such as surface plasmon resonance ,
conductivity, and redox behavior, leading to detectable signals. 

2
 OBJECTIVES

• To prepare Gold Colloidal Nanoparticles of size between 30 – 40 nm using Turkevich method.

• Cloning and Expression of S1 subunit recombinant His-tag proteins from pET-27 vector coupled with BL21 host with engineered T7 RNA polymerase.

• Purification of the obtained protein using IMAC (Immobilised Metal ion Affinity Chromatography).

• Bioconjugation of Colloidal gold nanoparticles with Recombinant protein.

SCOPE

• The development of Bioconjugated Gold Colloidal nanoparticles with S1 subunit recombinant protein.

• To study the possibility of developing a Rapid test kit for COVID-19 using Bioconjugated Colloidal Gold nanoparticles.

3
 METHODOLOGY

• SYNTHESIS OF NANOPARTICLES
• 1.0 mM HAuCl4 is prepared in 50 ml distilled water and is taken in a 250 ml RB flask.
• Its heated to 100˚C on heated stirrer.
• When it starts boiling 1.8 ml of 97 mM of Sodium Citrate is added through a condensing apparatus.
• Within 3-5 mins colour change changes from Yellow to Black to Wine Red.
• After colour change the solution is kept in constant heat for 10 mins.
4
• Centrifuge @ 7000 rpm for 15 min and discard pellet and store supernatant.
• EXPRESSION, PURIFICATION AND BIOCONJUGATION

The S1 subunit of COVID-19

pET Vector system IPAC Purification Bioconjugated Gold Colloidal nanoparticles

• Cloning and Expression of Recombinant His-Tag S1 subunit proteins using standard protocol.
• Inducing the desired protein using IPTG (isopropyl-β-D-thiogalactopyranoside), which induces the T7 polymerase, which in turn transcribes the target DNA in the
plasmid in large scale cultures.
• Using techniques like French Press and IPAC for lysing the cells and purifying the protein obtained, respectively.
• Confirming the purity of the using SDS-PAGE and confirmation of protein using Western Blot.
• Finally, conjugation of the Au Nanoparticles with the recombinant proteins obtained after purification. 5
 CHARACTERIZATION OF Au NANOPARTICLES

• The peak at 3307 cm-1 indicates C-H ring stretching

• The procurement of peak at 529nm using UV-Vis
vibrations whereas the peak at 1642 cm -1 is characteristic
Spectroscopy is indicative of formation of Gold
to N-H bending (or –C=C- bond) and the symmetric The formation of Gold Nanospheres can be seen in the
Nanoparticles.
component of the C-C (or C-H) stretching vibrations for above SEM image. Particle size ~ 30-40 nm.
citrate method of synthesis. A small bending peak near
1050 cm-1 indicates C-N bonding which is the citrate
molecule precursor.

6
 RESULTS

75 KDa

• SDS PAGE analysis of the lysed cells

• Western Blot of Recombinant S1 subunit
Lane 1: Supernatant after cell
lysis
Lane 2: Pellet after cell lysis Lane 1: Prestained Marker
• Expression study of (BL21(DE3)-pET27b- • SDS PAGE analysis of eluted rS1
Lane 2: S1 protein
Spike-1) colonies in different temperature
Lane 1: Induced BL21(DE3)-pET27b-S1
Lane 2: E1 - Imidazole eluted purified rS1
Lane 1: Protein Marker
Lane 2: Induced vector control (BL21(DE3)-pET27b) Lane 3: E2 - Imidazole eluted purified rS1
Lane 3: Uninduced colony 2 at 37 oC (BL21(DE3)-pET27b
Lane 4: Induced colony 2 at 37 oC (BL21(DE3)-pET27b
Lane 5: Induced colony 2 at 30 oC (BL21(DE3)-pET27b
Lane 6: Induced colony 3 at 37 oC (BL21(DE3)-pET27b
Lane 7: Induced colony 3 at 30 oC (BL21(DE3)-pET27b

• Bioconjugated Gold Colloid Nanoparticles

7
 CONCLUSION & FUTURE WORK

• Gold Nanoparticles have been synthesized successfully .

• But the project still has scope for further development.
• The method used for synthesising Au Nanoparticles can be optimized and better size control can be achieved in order to produce smaller
particles.
• The obtained Bioconjugate Gold Nanoparticles can be tested against real-time sera to check effectiveness of the protein affinity and Au’s
sensitivity.
• This can be further researched upon to provide a feasible, commercial Rapid Diagnostic Test kit.

8
 REFERENCES

• Turkevich, J., Stevenson, P. C., & Hillier, J. (1951). A study of the nucleation and growth processes in the synthesis of colloidal gold. Discussions
of the Faraday Society, 11, 55-75.

• Grabar, K. C., Freeman, R. G., Hommer, M. B., & Natan, M. J. (1995). Preparation and characterization of Au colloid monolayers. Analytical
chemistry, 67(4), 735-743.

• Kumar, S., Gandhi, K. S., & Kumar, R. (2007). Modeling of formation of gold nanoparticles by citrate method. Industrial & Engineering
Chemistry Research, 46(10), 3128-3136.

• Lee S. Y. (1996). High cell-density culture of Escherichia coli. Trends in biotechnology, 14(3), 98–105.

• Esposito, D., Mehalko, J., Drew, M., Snead, K., Wall, V., Taylor, T., Frank, P., Denson, J. P., Hong, M., Gulten, G., Sadtler, K., Messing, S., &
Gillette, W. (2020). Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays. Protein expression and
purification, 174, 105686.

• Rayavarapu, R. G., Petersen, W., Ungureanu, C., Post, J. N., van Leeuwen, T. G., & Manohar, S. (2007). Synthesis and bioconjugation of gold
nanoparticles as potential molecular probes for light-based imaging techniques. International journal of biomedical imaging, 2007, 29817.