Motive of this

research

 As far the researchers searched, there was no

colorimetric assay-based detection of selected
HbA1c aptamers with G@NPs is performed.
 Current improvements in low-cost, stable,
portable, specific and simple assays to detect
HbA1c are in great demand, facilitating the
monitoring of HbA1c in the blood for early
prognosis in diabetic patients
 The present detection has been based on
aptamers with G@NPs developed as a new
Objective
Binding of Thiol-modified aptamer
oligonucleotides containing gold
nanoparticles to glycated Hb with high
affinity.

To prepare gold nanoparticle-bound

purple aptamer aggregates that can
rapidly collapse into dispersed
nanoparticles upon binding of HbA1c

Detection of HbA1c in unpretreated whole

blood after dilution at pH 7.4
 Introduction
Diabetes mellitus , a Glycated hemoglobin is an
Glycation is an irreversible
chronic metabolic disorder, adduct of those
process leading to the
is one of the most consequences from the
production of HbA1c which
significant health problems non-enzymatic reaction of
can be used as a marker to
today. glucose with the N-
know the glucose status
terminal valine of
over the past three months
hemoglobin β-chains

Therefore, HbA1c is useful HbA1c analysis is a

to measure the level of powerful research tool and
glucose regulation the only retrospective
independent of daily indicator of glucose
glucose changes and regulation over time in
unaffected by recent diabetic patients
exercise or food intake
 Methodology
Preparation of aptamer-HbA1c modified G@NPs solution

40µl of Thiol modified 60bp ssDNA seq of HbA1c

Conjugated with 60µl of G@NPs by heating to 90°C and cooling continuously to room temperature.

This reaction results into the formation of HbA1c Aptamer.

The resulted HbA1c aptamer was mixed with 1mM of TCEP (different concentrations from 0.1µM to 100µM were mixed & stored at 28°C for 30
mins for disulphide cleavage to organize free thiol groups (-SH) of the G@NPs.

As a result, G@NPs wraps over the HbA1c aptamer with free organized thiol groups.

A parameter colorimetric test was done for the confirmation of the binding of G@NPs with HbA1c aptamer.
 Methodology

Time mapping The color After mixing of G@NPs & *Van der Waal forces Absorbance was
was done for 1 change was aptamers for few seconds, bound G@NPs-HbA1c measured with
min to 20 seen best at 7 using ultrasonicator, 7 mins aptamers are formed UV-VIS
mins. mins. interval. incubation was done at with monodisperse layer Spectrophotomet
room temperature. of G@NPs and acts as a
er in a 100µl
bridge between HbA1c
quartz cuvette.
target molecule and
HbA1c aptamer which
later causes aggregation.
 Results

Fig 1: TEM images of the nanoparticles obtained from the solutions containing Fig 2: The UV-vis spectra of Apt-
G@NPs (A) and HbA1c-G@NPs aptamers (B) HbA1c-G@NPs solution containing
0.1µg glycated hemoglobin at different
time but optimum time of assay was at
7 min
 Results
Specificity
This study concluded that uric acid, bovine
serum albumin, hemoglobin and glucose did not
specifically bind to HbA1c aptamer and thus
aggregation did not occur. However, in the
presence of HbA1c, G@NPs aggregated and
decreased absorbance at 525 nm.

Fig 2: The UV–vis spectra of Apt HbA1c-G@NPs

solution in the presence of different analytes
 Results
Reliability Repeatability & Reproducibility
Analytical recoveries of exogenously added To confirm the repeatability and reproducibility of
HbA1c (4% and 7%) in apparently healthy the method, the HbA1c levels in same blood
controls by the current assay were 94.0% samples were determined five times per day
and 95.4%, demonstrating the reliability of (within batch) and again after storage at 20°C for
the present colorimetric assay. one week (between batch) again they measured
the levels of HbA1c in same samples. The results
showed that determinations were almost
consistent indicating the good reproducibility and
reliability of the method.

Figure S3 UV–vis spectra of solution Apt-

HbA1c-G@NPs in the presence of different
concentrations (0.1µM to 100µM) of glycated
hemoglobin, Absorbance at 525 nm decreased or
shifted to higher wavelength slightly with
increasing concentration
 Results
Accuracy 12

HbA1c (%) by present method

10 f(x) = 0.959830866807611 x + 0.747357293868923
To confirm the accuracy of the current assay, R² = 0.989086412034562
8
HbA1c levels in blood samples were
measured by ELISA (x) and present method 6

(y). The HbA1c levels obtained by the current 4

assay matched with ELISA showing a good 2

correlation (r = 0.99, significant at 1% level) 0

5 6 7 8 9 10 11
HbA1c(%) by standared method

Figure S4: Correlation between whole blood

glycated hemoglobin values determined by
standard method (ELISA) and by present method
(Apt-HbA1c-G@NPs).
 HbA1c is a reliable method of
monitoring long-term diabetes mellitus

Conclusion control as it de- termines the average

blood glucose level of an individual
during a period of approximately 3
months.

Estimation of HbA1c is necessary to

follow up the treatment in DM as well
as it tells us need of adjustment of
dose to get blood sugar in normal
range.

So, the researchers proposed an

aptamer-based colorimetric assay of
HbA1c binding capacity using G@NP in
diluted blood samples to monitor long-
term glycemic control in diabetic
patients.
Thank You
 Materials and Reagents

 60 bp Thiol modified oligonucleotides ssDNA

sequence.
 Fixed 18 bp of ssDNA at the 3’ and 5’ ends.
 Purified HbA1c, BSA, Hemoglobin, Glucose.
 Sodium citrate
 Sodium tetrachlorocuprate- HAuCl4
 Uric Acid, Tris-(2-Carboxyethyl)Phosphine
hydrochloride