Development of aptasensors for malaria diagnosis using plasmodium falciparum glutamate dehydrogenase as target antigen

1. Development of aptasensors for malaria using Plasmodium
falciparum Glutamate dehydrogenase as target antigen.

2. Contents
 Chapter 1: Introduction, motivation and objectives
 Chapter 2: Cloning, expression, purification and characterization of
Plasmodium falciparum and Human, glutamate dehydrogenase.
 Chapter 3: Development of aptamer specific for PfGDH and its
characterization.
 Chapter 4: Protein induced fluorescence enhancement based biosensor
 Chapter 5: Development of capacitive aptasensor
 Chapter 6: Development of aptaFET biosensor
 Chapter 7: Dye coupled aptamer captured enzyme catalysed reaction for
detection of Pan specific and P. falciparum malaria in laboratory set up
and instrument free paper based format.
 Summary of work
 Future direction of research
 Publications
 References
 Acknowledgements

3. Chapter 1
Introduction

4. Introduction
Source WHO, NVBDCP,India

5. MDR Malaria: A Global threat
Plasmodium falciparum distribution
Drug resistant malaria
 RTS S –The world’s first malaria
vaccine by GSK and Gates
foundation
 Targeting sporozites
o Low efficacy
o Genetically variablilty

6. 6
Techniques Sensitivity
(parasites/µ
l)
Pros Cons
Microscopy 5-10 Distinguishes
between species
and stages, gold
standard
Requires
skilled labour
time
consuming
Quantitative
buffy coat
<15 Easy and
sensitive
Expensive and
chances of
leaking and
loss of
parasites
Polymerase
chain reaction
≥1 Highly sensitive Time and
resource
intensive
Rapid
diagnostic tests
(RDTs)
50–100 Low cost and fast
detection (~30
min)
Less sensitive,
non
quantitative
and unstable
RD
Ts
Diagnostic methods
Malaria Diagnosis

7. 7
Biomarkers for malaria
Lactate dehydrogenase (LDH)
-Structurally and kinetically distinct from human LDH, however not specific to
P.falciparum
Hemozoin
-Appears as clusters in the digestive vacuole, Not detectable in young ring
stages
Glutamate dehydrogenase (GDH)
-Absent in RBC. Present in low concentration
Aldolase
-Distinct from host Aldolase, only detectable in high concentration
Histidine rich protein –II (HRP-II)
-Unique biomarker specific to P.falciparum, False negatives due to Prozone effect
The current available most of RDTs in
the market based on Targeting of
PLDH as Pan specific and PfHRP-II as
falciparum biomarker.

8. Biomarkers for Malaria
• Just enough data to raise suspicion
• WHO-Recommendations against use of
HRP-II based RDTs.

9. Pf GDH is potent biomarker for Malaria
Location
Sequence
Structure
Expression through out the parasitic life
cycle
Present in good concentration in parasitic
cytoplasm
Specific Inhibitor for PfGDH
Presence in serum
18 December 2022 9

10. 10
Challenges
Quantitative test: Quantitatively estimate the
biomarker
Stability: Antibody based systems are unstable
in hot and humid climates
Sensitivity : Efficient to detect biomarker in
case of asymptomatic malaria
Low cost: Especially with the point of view of
developing countries

11. 11
Chapter 2
Cloning, expression, purification and
characterization of Plasmodium
falciparum and human glutamate
dehydrogenase

12. Cloning, expression and purification of PfGDH and HGDH
• cDNA from 3D7 asynchronous P. falciparum Obtained from ATCC, HGDH TA clone obtained
from Bioserve.
• 0.8 % agarose gel stained with EtBr, 10 % SDS PAGE stained with commassie blue stain.

13. Characterization of PfGDH and HGDH
Western blot of recombinant PfGDH (I) and HGDHa (II) with anti His antibody (1:5000
dilution) over PVDF membrane (A). Secondary structure estimation of PfGDH and HGDHa
(1mg/ml) in 10 mM phosphate and Tris buffer respectively (B).
Alpha Beta Turns Random
PfGDH 28% 5% 30% 37%
HGDH 27% 58% - 15%

14. Characterization of PfGDH
C
• AUC result of PfGDH showing
different fractions of oligomers
(A, B).
• Maldi Mass Spectroscopy Analysis
of PfGDH, hexameric protein of
PfGDH denatured in the presence
of 0.5M DTT and 10% SDS.
~54 KDa

15. Enzyme Kinetics parameters for PfGDH
18 December 2022 15
A. PfGDH activity was measured spectrophotometrically by following the change in absorbance at 340
nm. The forward reaction (deamination of glutamate) was followed in the presence of 100 µM NADP
in assay buffer (50 mM potassium phosphate buffer), pH 8.0.
B. Competitive Inhibition of PfGDH in presence of Isopthalic Acid. (Deamination of glutamate) was
followed in the presence of 100 µM NADP in assay buffer (50 mM potassium phosphate buffer),
pH 8.0, Glutamate- 10mM.

16. Conclusion
• The PfGDH, HGDH were cloned, expressed and purified.
• The Purified protein characterized through various techniques (AUC,
CD Spectroscopy, MALDI-MS).
• The Michaelis Menten (Km) value is calculated for recombinant
PfGDH, HGDH and found 4.10±0.28 mM, 4.36±0.18 mM respectively.
• Isopathalic acid act as specific inhibitor to competitively inhibit the
PfGDH activity not HGDH.
16

17. 17
Chapter 3
Development of aptamer specific for
PfGDH and its characterization
Ready to bind
with Target

18. AFM images of A. Bare PVDF membrane.
B. PVDF-PfGDH. C. PVDF-PfGDH Aptamer.
Library
5’-CACCTAATACGACTCACTATAGCGGATCCGA-N40-
CTGGCTCGAACAAGCTTGC-3’
Forward primer:
5’-CACCTAATACGACTCACTATAGCGGA-3’
Reverse primer:
5’-Biotin-GCAAGCTTGTTCGAGCCAG-3’
18
Decreasing Binding time
PCR amplification at the end of SELEX
cycle 1-17 respectively. Counter selex against
PVDF after 6, 8, 10 (Marked with Yellow
Star) and Counter selex against HGDH after
S13, S15 (Marked with Red Triangle).
Screening and characterization of aptamers specific for PfGDH

19. SELEX- Cloning and Sequencing
19
Screening of 55 TA clones
through restriction enzyme
(EcoR1) digestion. M: marker,
Control: digestion product of
plasmid from a blue colony.
Positive clones are labeled
with star selected for
sequencing

20. SELEX- Cloning and Sequencing
20
Selex Alignment- Different aptamer sequence group on the basis of percentage sequence matches in
decreasing order (A>B>C>D>E)
Group Aptamer
Name
Sequence of Aptamer Percentage
sequence
match
A NG37
NG46
NG51
NG53
TCCGACCACGGCCAGCTCACCACGCTCCTCCCCCCCTCCCCCGC
----------CCCCACTCACTGCTCGGACTCTCCCCCTCCCCGC
----------CCCCACTCACTGCTCGGACTCTCCCCCTCCCCGC
----------CCCCACTCACTGCTCGGACTCTCCCCCTCCCCGC
B NG3
NG55
NG9
NG36
TCCGAGCCGGGGTGTTCTGTTGGCGGGGGCGG--TGGGCGGG---
TCCGAGTGGCCTGGGCGTGCGGGGGTGGGGGGGTGGAGCGCGGC-
TCCGAGTGGCCTGGGCGTGCAGGGGTGGGGGGGGTGGAGCCTGGC
TCCGAGTGGCCTGGGCGTGCGGGGGTGGGGGGGTGGAGCGCGGC-
C NG4
NG15
NG11
TCCGATTCCCCCCCCGCACCTCACCCTACCATGCCCGCCTCCCGC
TCCGACCCCCCCTCCTGTGCTCGTCGTG-GCTCTTCCATTCCGGC
TCCGAACCCCACGAAGTGTGCTGGCTCTT-CTTGACCCATTCCGGC
D NG24
NG29
NG18
TCCGACACCCGGGTTTTCCCATCCGTTCCCCCGCTCCCCCCGGC-
TCCGAGGGG-GGGTGGGTGGTTCCCTCCCGCCGCTCCCCCTTGGC
-------CG-TGATGCTCTCTCTCCTCCCGCCGCTCCCCCTTGGC
E NG21
NG35
TCCGACAACAGCCCACCCCCCACCCCGGACAACTCCCTGCT-----CGGC
TCCGACCTATC-----CCCCTTCCCCGGTTCTCCTTCTGGTCTCGTCGGC
Decreasing
order
of
percentage
sequence
matches

21. Structure prediction of aptamer NG3 and NG51
21
• The sequences of NG3 and NG51 uncovers the presence of G-rich regions which
might contribute to the formation of complex G-quadruplex structures.
• The G quadruplex prediction software QGRS Mapper predicted a total of 2 QGRS
sequences for NG3 and Zero QGRS sequence for NG51.
A. CD analysis of NG3 and NG51 aptamer. (B)Secondary structures of NG3 and NG51 aptamer
predicted by mfold software.
NG NG3
NG51
Signature peaks of
Anti Parallel G quadruplex

22. Electrophoretic mobility shift assay with NG3 and NG51
22
Electrophoretic mobility shift assay (EMSA). PfGDH at various concentrations (0.1uM to 2.4uM,
and 0.25 nM Aptamer NG3, NG51. Stained with cyber gold and Commassie blue staining in 6%
polyacrylamide native gel.

23. Binding affinity of developed Aptamer in solution
23
CD spectra (A) NG3 and (B) NG51 in the presence of the target protein PfGDHa (0µM to 1.0µM) and negative control
proteins HGDHa, HSA( Human Serum Albumin), HRP-II (Histidine rich protein), PfLDH (Plasmodium falciparum
lactate dehydrogenase) CD spectra of (C) NG3 and (D) NG51.
Kd for NG3=0.5 ± 0.04 μM
Kd for NG51=1.1 ± 0.20uM
F= Bmax* abs(x)/Kd+abs(x)

24. Binding affinity of developed Aptamer on surface
24
SPR data of NG3 (A) and NG51 (B) with different concentration of PfGDH and dissociation
constant (Kd) for respective aptamer . Specificity of aptamer with analogous protein.

25. Conclusion
• The two aptamer (NG3, NG51) against PfGDH were developed.
• The NG3 forming the G quadruplex structure.
• The Kd found 0.5±0.04 µM and 1.1± 0.2 µM with CD spectroscopy
for NG3 and NG51 respectively.
• The Kd found 78±4.58 nM and 370±14.37 nM with SPR spectroscopy
for NG3 and NG51 respectively.
• Gibbs Energy was also calculated for both of aptamer (NG3, NG51)
and found -56.9, -36.65 KJ/Mole respectivily based on SPR data
indicate prompt interaction of aptamer with target.
25

26. 26
Chapter 4
Protein induced fluorescence
enhancement based detection of PfGDH
using aptamer carbon dot.
Handheld fluorescence spectrophotometer

27. Fluorescence based Detection of PfGDH
FRET is sensitive, but a tough technique to implement.

28. C Dots synthesis and characterizations
𝐷
𝐺
= 1.03

29. Covalent linking of Aptamer with C Dots
FTIR spectra of NG3aptamer (black) Cdot (blue) and Cdot-aptamer conjugate after EDC/NHS
activation covalent linkage (red).

30. Fluorometric Detection of PfGDH for Malaria
(A) Excitation and Emission spectra of Cdot, aptamer, PfGDH, Cdot-aptamer and Cdot-
aptamer/PfGDH complex in binding buffer. (B) PIFE of Cdot-aptamer conjugate with increasing
concentration of PfGDH protein spiked in binding buffer (C) Calibration plot for PIFE response
with PfGDH protein spiked in binding buffer
QY of Cdot/apt 34%
Increased up to 40 %
in presence of PfGDH

31. Fluorometric Detection of PfGDH for Malaria
A. Time optimization study for PIFE. B. PIFE response in srum. Calibration plot for PIFE response of the Cdot-
aptamer conjugate against different concentration of PfGDH spiked in human serum diluted in binding buffer
(E) PIFE response of Cdot-aptmer conjugate with different nonspecific malaria (PfLDH, PfHRP-II) and human
(HSA, HGDH) proteins each at a concentration of 10nM spiked in binding buffer.
LOD is found 0.48 and 2.58 nM in
buffer and serum respectively.

32. Docking of PfGDH with NG3 aptamer
• NG3 Sequence mFOLD Dot bracket annotation of NG3 3D structure of
NG3 Dock with PfGDH(2BMA) and NG3 with patchdock/PLIP software
Analysis through PyMOL.
32
Name of amino acid and position
Hydropho
bic Pocket
Meth 384, Gly 300, 370,169 Leu 119, Lys 401, Try 195,
Ile 397,281 Cystine 446, Arg 395, Glu 444,440
Interacting
Amino
acid/
Nucleotide
Hydrophobic
Interaction
H- Bond Salt Bridge
299Ser:28C
313Pro:81C
396Glu:56C
41Lys:49C
203Asn:53G
205Arg:45G
211Tyr:43C
440Glu:45G
41Lys:48G
205Arg:45,46T
255Lys:83T
274His:43,67C
Florescence life time of Cdot, Cdot-aptamer and
Cdot-aptamer-PfGDH with excitation at λ 308nm.

33. Conclusion
• Carbon dot of average size ~ 2nm were synthesized from L glutamic
acid through pyrolysis.
• Amine modified Aptamer covalently linked with C dot through
EDC/NHS chemistry.
• Selective protein induced fluorescence enhancement were observed
in Cdot/Aptamer for various concentration of PfGDH (1nM to 50nM)
no interference from analogous protein and in serum .
• LOD is found 0.48, 2.58nM in spiked Buffer and Serum (4x diluted)
respectively.
33

34. 34
Chapter 5
Capacitive Aptasensor based detection of
PfGDH for Malaria diagnosis

35. Capacitive Aptasensor based detection of PfGDH for
Malaria diagnosis
35
 For POC application Non faradic EIS is preferable than faradic EIS
Cdl
Csl

36. Model of Capacitive response
.
36

37. Characterization of SAM
37
The results of AFM characterization before and after modification showed that the topographic roughness
gradually decreased from bare gold (1.52 nm) to aptamer modified gold electrode (1.41 nm) and blocked
aptasensor electrode (1.33 nm). The cyclic voltammetry performed in presence of K3(Fe(CN)6).

38. Detection of PfGDH in spiked undiluted serum
38
(A) Non-Faradaic measurement data in serum Phase response vs frequency. (B) Capacitance
response of NG3 aptamer-PfGDH with circled enlarged image in inset. (C) Calibration curve plot
capacitance response at 2 Hz. (D) Response of NG3 aptamer with analogue proteins spiked in
serum

39. Conclusion
• Surface assembled monolayer successfully formed with NG3
aptamer/MCH in 1:100 ratio .
• The Sensor has been developed which can detect PfGDH in buffer
and undiluted human serum in linear range (100fM-100nM).
• The sensitivity of the aptasensor as derived from the slopes of the
calibration curves in serum and buffer samples were 0.12 (10-7F)/ log
([PfGDH] pM) and 0.09 (10-7F)/ log([PfGDH] pM), respectively
• The limit of detection was calculated 0.43±0.08pM, 0.77±0.13pM for
buffer and serum respectively.
39

40. 40
Chapter 6
Development of aptamer based field
effect transistor (aptaFET) biosensor for
detection of PfGDH in serum sample.

41. Extended Gate Field Effect Transistor for Malaria Diagnosis
41
In EGFET, Gate is extended from transistor

42. Working circuit and IDµE
42
Working circuit of aptaFET biosensor. An interdigitated
gold electrode (IDµE) with pseudo reference electrode
connected to the gate of n type MOSFET. The aptamer
target binding reaction carried out on surface of IDµE
and FET system transduced this binding event into
electrical signal.

43. Characterization of SAM
43
A. Characterization of surface modification with AFM, (i) bare gold chip electrode (ii) surface assembled monolayer with
MCH/Aptamer/ Au. (B) Faradic EIS characterization of sensor fabrication, over IDµE chip and scanned in 10 mM potassium
hexacyano ferrate solution prepared in PBS. (C) Stability studies of IDµE in binding buffer from the plot of drain current
versus time.
S.N
o
Electrodes/SAM Rs (Ω) Rct (Ω) Cdl (µF) W (Ωs-1/2)
1. IDµE 144.27 118.5 17.1 885.66
2. IDµE/Aptamer/MCH 151.14 620.23 2.56 848.74
3. IDµE/Aptamer/MCH/Blockin
g
155.76 1778.1 1.34 825.34
Rms roughness 0.41
Average height 1.35
Rms roughness 0.53
Average height 1.48
Rms roughness 0.47
Average height 1.71
Theoretically calculated Debye length (ƛ)= 1.5

44. Working principle of FET for Sensor applications
44

45. Detection of PfGDH in spiked buffer and diluted serum
45
A) FET response of PfGDH spiked in buffer, with (B) calibration plot between PfGDH
concentration in log10 term (0.1pM to 10nM) and shift in Vgs. (C) FET response of PfGDH spiked
in serum, with (D) calibration plot of PfGDH concentration verses shift in Vgs
LOD was found 16.7 and 48.6 pM in spiked
buffer and 10 fold diluted serum
Calculated Isoelectric point (pI) of PfGDH= 6.6
Theoretically calculated pI for PfGDH = 7.4

46. Interference study of EgFET sensor
46
A. Plot of Id versus Vg voltage for the target (PfGDH) and different potential interfering
proteins. The analysis was performed in buffer at 10nM concentration with 30 min time of
incubation, shift in Vg observed at 1.4 µA drain current. (B) Specificity of NG3 aptasensor
with analogous spiked protein

47. Conclusion
• Surface assembled monolayer was formed over Interdigitated
micro gold electrode and characterized.
• The EgFET Sensor has been developed which can detect
PfGDH in buffer and diluted human serum in linear range
(100fM-10nM) respectively.
• The Limit of detection was calculated and found 16.7±
1.88pM, 48.6± 3.47pM for buffer and serum respectively.
47

48. 48
Chapter 7
Dye coupled aptamer captured enzyme catalysed reaction
for detection of Pan specific and P. falciparum malaria in
laboratory set up and instrument free paper based format.

49. Dual Colorimetric/fluorometric Based Malaria Diagnosis
49

50. Resazurin/Resorufin
• Resazurin non toxic, cell permeable and non fluorescent blue colour
compound.
• Resazurin reactivity with NADH and other reducing agents transform
into Resorufin which is highly fluorescent, pink colour
50

51. Optimization of Aptamer/Protein loading with Mag Beds
51
Binding ratio of aptamers with enzyme capture efficiency with streptavidin coated magnetic
beads for (A) NG3/PfGDH and (B) P38/PLDH. Time optimization study for absorbance intensity at
different concentrations of the target captured enzymes (C) PfGDH (D) PLDH.

52. Detection of Malaria Biomarker PfGDH, PLDH with
Instruments
52
Calibration plots derived from absorbance and fluorescence characteristics for (A) PLDH, (B)
PfGDH in binding buffer and (C) PLDH, (D) PfGDH in serum.

53. Syringe Modification and Operation
53
Fabrication of modified syringe (A) real image with its components (B) Drawings of fabricated
syringe.

54. Detection of Malaria Biomarker PfGDH, PLDH without Instrument
54

55. 55
Paper based analytical devices
 Rapid analysis
 Less expensive
 Small sample volume
 Little or no external
equipment or power
Affordable
Sensitive
Specific
User friendly
Rapid-Robust Equipment
free
Delivered to those
who need it
ASSURED ( WHO)
(Martinez et al, 2010 Diagnostics for the developing world: microfluidic paper-based analytical devices )

56. Preparation of polymer coated paper
56
SEM images showing surface topography of modified paper (A), control (i), chitosan treated (ii),
sericine treated (iii) and DEAE treated (iv) at 250 x magnification and inset respective image
magnified at 5000 x. Dye entrapment efficiency over Whatman filter paper (control) and
modified paper (B). The pixel intensity and Overall dye adsorbing efficiency by treated paper.

57. 57
Detection of Malaria Biomarker PfGDH, PLDH without
Instrument
Colour response on paper matrix against different concentration of PLDH (A), PfGDH (B) spiked
in binding buffer and PLDH (C), PfGDH (D) spiked in serum.

58. Detection of biomarker PfGDH+PLDH and specificity spiked
in Buffer
58

59. Conclusion
59
• Ratio of Aptamer (NG3, P38) with streptavidin magnetic beads
optimized and found that 1:20 ratio showed maximum loading.
• Optimization of time have been performed and found 60 min suitable
for diagnostic test*.
• Syringe with magnet and paper wick has been modified .
Technique Surface/
Electrode
Probe Detection
range
Target
Antigen
Response time LOD
Instrument based
UV-Visible
spectroscopy
Magnetic
beads
aptamer 1 pM- 100
nM
PLDH and
PfGDH
60 min
0.55±0.09 pM,
1.34±0.12 pM for
PLDH and PfGDH
respectively
Instrument based
Fluorescence
spectroscopy
1.72±0.13 pM,
1.43±0.14 pM for
PLDH and PfGDH
respectively
Instrument free
68.75 ± 7.64 ,69.25
± 8.22 pM for
PfGDH and PLDH
respectively
For Instrument based LOD = 3X SD of Blank/ slope of calibration
curve
For Instrument free LOD = LOB + 1.65 x SD of lowest concentration,
where limit of blank (LOB) = mean of blank + 1.65 x SD of blank

60. Overview of work
• PfGDH and HGDH genes were cloned, Expressed, Purified and characterized.
• Aptamer for PfGDH has been developed through SELEX procedure. The dissociation
constant was measured in liquid (CD) and over solid surface (SPR).
60
S. No Method Platform Range of Detection Limit of Detction
1. Carbon Dot based Fluorescence
Spectroscopy
1 to 50nM 2.58nM in Diluted Serum (4X)
2. Capacitive aptamer based Non faradic
electrochemical
spectroscopy
100fM to 100nM 0.77pM in Serum
3. Aptamer Based EgFET Non faradic FET
sensor
100fM to 10nM 48.6pM in Diluted Serum
(10X)
4. Magnetic beads based
detection (DECAMAL) for
PfGDH and PfLDH.
Colorimetric,
Fluorimetric,
Paper based
1pM to 100nM 0.55 (PLDH), 1.34 (PfGDH) pM UV-
Vis in Serum
1.72 (PLDH), 1.43 (PfGDH) pM
Fluorescence in Serum
110 (PLDH), 121 (PfGDH)
Instrument free in Serum

61. 61
Future direction of work
 The three dimension structure for PfGDH is already known, to understand exact
mechanism and binding motif of developed aptamer over PfGDH, interactions
study through X-ray crystallographic, NMR spectroscopy need to be done in
order to decipher the linkage chemistry.
 The capacitance based sensor concept could be executed in a pragmatic lab on
chip platform similar to glucose sensor for fast, simple malaria detection
approach. Since the above sensor performed at very low frequency (2 Hz),
designing of a miniaturized instrumentation for point of care device is easy for
implementation.
 In dye based method to further reduce the cost SMB can be replaced with
chitosan capped iron oxide magnetic particles and functionalised with aptamer
to fulfil current market need for low cost (~0. 20 $ per test) malaria.

62. S.
No.
Name Sequence of Aptamer (5'-3') Target
1 P38 5'(Biotin)TTTTCACCTAATACGACTCACTATAGCGG
ATCCGACAATAATACACTTTGCTCCCCTGTGGCTTT
TCGCACTCGCCTGGCTCGAACAAGCTTGC-3'
PLDH
2 NG3 5'(Biotin)TTTTCACCTCATACGACTCACTATAGCGG
ATCCGAGCCGGGGTGTTCTGTTGGCGGGGGCGGTG
GGCGGGCTGGCTCGAACAAGCTTGC-3'
PfGDH
S. No Name Buffer composition
1. Coupling Buffer 10 mM TrisCl, 100 mM NaCl, pH 7.5,
2. Binding Buffer 50 mM potassium phosphate buffer, 5 mM NaCl, 5
mM KCl, 2.5 mM MgCl2 , pH 8.0
3. Cocktail Buffer Lactate-50 mM, Glutamate-10 mM, APAD-1 mM,
NADP-0.2 mM, Resazurin-0.40 mM, Phenazinetho
sulphate- 0.37 mM in 50 mM potassium phosphate
buffer

63. Improvement of Aptamer Affinity by Dimerization, February 2008, Sensors 8(2)
DOI: 10.3390/s8021090

64. The colloidal milk powder was used as reference solution. The average life time was calculated
from equation (ii). Where Ai is distribution of particle and τ is measured life time.