Fluorescence immunosensor for cardiac troponin T based on F?rster resonance energy transfer (FRET) between carbon dot and MoS2 nano-couple

Article abstract of DOI:10.1039/c8cp02433b

In this study, we demonstrated a prompt and sensitive detection technique for cardiac troponin T (cTnT) in buffer and biological fluid (serum) using an NIR-active fluorescent anti-cTnT-labelled carbon dot (CD) and molybdenum disulfide (MoS₂)-based nano-couple. Exfoliated MoS₂ nanosheets strongly grasp the anti-cTnT-labelled CDs over their surface, and an excited-state non-radiative energy transfer mechanism takes place from CDs to MoS₂, thereby quenching the upconversion fluorescence. The nonlinear and upward Stern-Volmer relationship is observed, which indicates a combined static and dynamic quenching. Static and time-resolved fluorescence measurements predict distance-dependent F?rster resonance energy transfer (FRET) dynamics, which control the detection process. In the presence of cTnT, the energy transfer process gets hindered due to strong antibody/antigen (anti-cTnT/cTnT) interaction. The cTnT molecules affect the positions of the nano-couple and cause effective detachment of CDs from the MoS₂ surface. This results hindrance in the energy transfer process with consequent restoration of upconversion intensity. A linear response is observed between the cTnT concentration and the restored fluorescence intensity in the concentration range of 0.1-50 ng mL^-1 with a limit of detection of 0.12 ng mL^-1 and a limit of quantification of 0.38 ng mL^-1. Statistical analysis shows that the present assay possesses an accuracy of 101.4 ± 3.76 with a co-relation co-efficient of 0.99. Thus, CD/MoS₂ provides a promising platform for the sensitive detection of cTnT.

Full text of DOI:10.1039/c8cp02433b

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Förster Resonance Energy Transfer (FRET) Between Carbon
Dots/MoS2 Based Fluorescence Immunosensor for Sensitive
Detection of Cardiac Troponin T
Received 00th January 20xx,
Accepted 00th January 20xx
Satyabrat Gogoi* and Raju Khan*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this study, we demonstrate a prompt and sensitive detection technique of cardiac troponin T (cTnT) in buffer and
biological fluid (serum) using NIR active fluorescent anti-cTnT labelled carbon dots (CDs) and molybdenum disulfide (MoS2)
based nano-couple. Exfoliated MoS2 nanosheets strongly holds the anti-cTnT labelled CDs over its surface constituting an
excited state non-radiative energy transfer mechanism from CDs to MoS2 and thereby quenching the upconversion
fluorescence. Non-linear and upward Stern-Volmer relationship is observed which indicates a combined static and
dynamic quenching. Static and time resolved fluorescence measurements predict a distance dependent Förster resonance
energy transfer (FRET) dynamics which control the detection process. In presence of cTnT energy transfer process gets
hindered courtesy to strong antibody/antigen (anti-cTnT/cTnT) interaction. cTnT molecules effect the positions of the
nano-couple and causes effective detachment of CDs from MoS2 surface. Such effect of cTnT causes hindrance in energy
transfer process with consequence restoring of upconversion intensity. A linear response is observed between cTnT
concentration and restored fluoresence intensity in the concentration range of 0.1-50 ng mL-1 with limit of detection 0.12
ng mL-1 and limit of quantification 0.38 ng mL-1. Statistical analysis shows that current assay posseses an accuracy of
101.4±3.76 with co-relation co-efficient 0.99. Thus, CDs/MoS2 provides a promising platform for sensitive detection of
cTnT.
before coronary artery stenosis. Early release of cTn just after
the onset of AMI and their long persistence in blood (3.5 hours
up to 10 days) endorse them as preferred markers in the
Introduction
Acute myocardial infarction (AMI) has been identified as one
of the leading causes of morbidity and mortality globally [1, 2].
The major challenge imparting complications in the prompt
treatment of AMI is its time critical nature. Myocardial
infarction may occur with little or no history of symptoms [3].
Therefore, it appears that evidence based rapid and accurate
diagnosis plays the most significant role in AMI medical
management and treatment [4, 5]. An early detection of AMI
aids a rapid decision making and ensures the administration of
proper treatment to facilitate a better patient care [6].
detection of AMI related diseases [12]. However, despite
global acceptance of cTn as AMI risk marker, only one out of
three laboratories is following these assays [13]. This gives an
impression about the criticality of the currently available cTn
detection techniques. Generally, clinical methodology urges
for shorter turnaround time which operates with high
sensitivity. Most of the point of care (PoC) level techniques is
relatively complex and expensive as they work on integrated
microfluidic or capillary device which performs in multiple
cycles of sample loading and washing [14, 15]. In addition, an
ample quantity of sample is required which is collected
intravenously. These factors limit the scope of these
techniques and insist to search rapid, reliable, cost-effective
diagnosis of cardiac cTn.
In diagnostic and prognostic treatment of acute coronary
syndrome, human cardiac troponins (cTn) including troponin I
and T (cTnI and cTnT) have been recognized as the most
sensitive and specific biomarkers [7-9]. On the occurrence of
cardiac injury, cTn is released into the bloodstream [10, 11].
The presence of cTn is recognizable during heart failure even
Dealing with the challenges, nanotechnology based
immunoassays have gained tremendous interest in recent
years by exploring the useful properties of nanomaterials.
State of the art cites utilization of gold nanoparticle, single wall
carbon nanotube, TiO2 nanotubes, graphene and graphene
quantum dots etc. in the detection of cTn by utilizing their
optical properties [15-19]. In the domain of fluorescence based
detection techniques, excited-state energy transfer between
zero and two dimensional nanostructures (0D and 2D) have
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shown lots of promises [20-22]. Colloidal quantum dots (0D) CDs were prepared by a hydrothermal method by using 2.0 g
have been recognized as excellent absorbers and emitters of of starch soluble and 1.0 g of citric acid as the precursors [29].
light with high quantum efficiency. However, they possess The carbonization reaction was carried out in a Teflon lined
poor carrier transport properties. In contrast, 2D autoclave for 5 h at the temperature of 180 ⁰C inside an oven.
nanomaterials (graphitic materials and transition metal Prepared CDs were purified by subjecting into centrifuge
dichalcogenide (TMDC)) possess high mobility [23]. Thus, (503×g) followed by membrane dialysis (cut-off molecular
combination of these two classes of materials together seems weight of 300 Da). Two dimensional MoS2 NS were obtained
to constitute a unique proposition towards the development by exfoliation of bulk MoS2 following sono-chemical method
of novel sensing platforms. Taking the subject one step [30]. Bulk MoS2 was subjected into ultra-sonication for 48 h. It
forward, we would like to highlight carbon dots/molybdenum was then centrifuged at (503×g) to obtain a stable dispersion
disulfide (CDs/MoS2) nano-couple as
a potential Förster of exfoliated MoS2 NS.
resonance energy transfer (FRET) based sensing platform. CDs Labelling of CDs with anti-cTnT
are graphitic nanostructure which possesses the key optical
Labelling of CDs with anti-cTnT was done by EDC/sulfo-NHS
properties like broad UV absorption, stable emission (generally
in green and blue region), upconversion emission (activated by
NIR light) with other inherent advantages like non-toxicity,
water solubility, multi-functionality and facile preparation [24,
25]. CDs have been utilized in bio-imaging, cell-labelling and
sensing applications [25, 26]. Similarly, 2D single or few
layered TMDC nanosheets (MoS2/WS2 NS etc.) have attracted
extensive attention on account of their unique physical and
chemical properties. Compared to other 2D materials like
graphene, MoS2 possesses the advantage of non-toxicity [27].
Zhang and co-workers in 2013 reported first successful use of
MoS2 nanosheets for biosensing application [28]. Since then, it
has been a continuous process to utilize MoS2 to design novel
sensing platforms.
coupling method [31]. Briefly, antibody was activated by
incubating 990 μL of anti-cTnT with 10 μL of EDC-NHS solution
at room temperature with constant stirring for 15 min. The
solution of EDC-NHS cross-linker was obtained by adding 0.4
mg of EDC and 1.1 mg of NHS in 100 µL of 0.1 M of MES buffer.
Activated anti-cTnT was then incubated with CDs solution
(concentration of 0.05 mg mL-1) (at 1:1 v/v ratio) for 1 h to get
anti-cTnT functionalized CDs (anti-cTnT-f-CDs). Free
unconjugated molecules were separated by ultracentrifugation
method and membrane dialysis method.
Characterization and instrumentation techniques
Fourier Transform Infrared (FTIR) spectrometer, PerkinElmer
(USA), Model: Spectrum 100 and UV-visible spectrometer
(SPECORD-200, Analytic Jena AG, Germany) were used to study
the functional groups and absorption spectrum of the
nanostructures. X-ray Diffraction (XRD) patterns were
recorded by a JEOL, Japan, Model: JDX-IIP3A, diffractometer in
the scanning range of 2θ=10°-70° and at a scanning rate of 2°
min-1. Morphological attributes and elemental composition
were studied with the help of a field emission scanning
microscope (FE-SEM) of Zeiss Sigma VP, Germany, equipped
with Oxford Electron Diffracting Scattered (EDS) sputtering
system. In depth morphological study including size, shape and
size distribution was carried out by using High Resolution
Transmission Electron Microscopic (HR-TEM) analysis. Carbon
coated copper grids of TED PELLA INC, Ultrathin C, Type A, 400
mesh were used to visualize the particles under JEOL, JEMCXII,
Japan, microscope at an operating voltage of 200 kV.
Fluorolog3, Jobin Yvon, Edision, USA equipped with
Hamamatsu R928, NanoLED and DeltaHUB was used for static
and dynamic types of fluorescence measurements.
Considering these facts, the present study aspires to
advocate a NIR switching CDs/MoS2 based FRET couple for
highly sensitive detection of cTnT in biological fluid. The
objective to use NIR source is to enjoy benefits like biological
transparency and in depth tissue penetration which otherwise
difficult to achieve with UV-visible light [25]. Biological tissues
possess poor absorption and scattering of NIR light which
enhance signal to back ground ratio. NIR based probe can be
used at low working concentration which minimizes
concentration originated in vivo toxicity [25]. Thus, content of
the work includes in depth optical properties of CDs/MoS2
couple and their utilization in the detection of cTnT both in
buffer and serum.
Experimental
Materials
Starch soluble, citric acid, molybdenum disulfide (MoS2), N-
hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
other chemicals used were purchased from Sigma-Aldrich,
USA. Cardiac troponin T (cTnT) and troponin T antibody (anti-
cTnT) were obtained from Abcam, Cambridge, MA, USA. The
human blood serum was provided by Clinical Centre, CSIR-
NEIST, Jorhat. The sample was collected by using an
anticoagulant called acid–citrose–dextrose (Sigma-Aldrich,
USA).
Detection of cTnT in buffer and biological fluid (serum)
A fixed couple of anti-cTnT-f-CDs/MoS2 was used as a FRET
platform for the detection of cTnT maintaining a pH of 7.4. NIR
upconversion fluorescence spectra of anti-cTnT-f-CDs were
recorded at the excitation wavelength of 940 nm. Then a
nano-couple was constructed by adding optimized amount of
MoS2 NS. A varying amount MoS2 (5-50 µM) was used to
quench the fluorescence of anti-cTnT-f-CDs and a curve was
plotted to corroborate the relationship between the quenched
fluorescence intensity and concentration of MoS2 NS. The
curve gives the threshold concentration of MoS2 NS that bring
effective quenching of anti-cTnT-f-CDs. This optimized amount
Preparation of CDs and MoS2 NS
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of MoS2 NS was added to the working anti-cTnT-f-CDs solution. CDs reveals the characteristics surface plasmon resonance
Then, a varying amount of cTnT was added to the working peaks at wavelengths of 245 nm (appears as a shoulder) and
solution in the concentration range of 0.1-100 ng mL-1. All the 280 nm which were assigned as π- π* and n-π* transitions,
samples were incubated at 25 ⁰C for 15 minutes and their respectively (Figure 1.a). Similarly, MoS2 displayed
a
upconversion fluorescence spectra were recorded at the substantial absorption over a wide range of wavelength. Two
excitation wavelength of 940 nm. An enhancement in the prominent peaks can be phase out at the wavelengths of 617
upconversion intensity was expected. To validate
a co- and 679 nm, which were assigned as the peaks arising from
relationship between the enhanced fluorescence intensity and direct gap transitions at the K point of the Brillouin zone
cTnT concentration, a curve fitting procedure was adopted by (Figure 1.b). The absorption spectrum confirms light
using Origin 8.5 Pro analytical software. Validation of the absorption capability of MoS2 NS in the wavelength region of
detection process was carried out statistically by using 500-600 nm. The characteristic functional groups of CDs (3200
Microsoft Office Excel, 2010 (detail protocol is included in cm-1 for -O-H stretching, 2990 cm-1 for –C-H stretching, 1650
supporting information). All the fluorescence measurements cm-1 -C=O stretching and 1540 cm-1 for -O-C-O- stretching) are
were recorded at 25 ⁰C under similar environment with confirmed from the respective FTIR spectrum (Figure S1). XRD
maximum possible care. Dynamic fluorescence measurements pattern confirmed the crystal phase of MoS2 NS (Figure 1.c).
were further carried out to understand the energy transfer They belong to space group P63/mmc where six peaks can be
process. Detection of cTnT was also carried out in diluted identified at diffraction angles (2θ) of 14.8⁰, 32.7⁰, 33.7⁰,
human blood serum following the same procedure.
36.1⁰, 39.8⁰, 44.2⁰, 50.0⁰, 58.5⁰ and 60.7⁰. These peaks are
assigned as (002), (100), (101), (102), (103), (006/104), (105),
(110) and (008/112) plane of nano MoS2 phase.
Crystallographic data were confirmed by comparing with the
data obtained from ICDD-PDF-2 (Card no-77-1716). In contrary
to MoS2 NS, CDs displayed a broad peak near 2θ value of 21⁰.
This peak is assigned as the weakly crystalline graphitic carbon
peak. The carbon phase in CDs may be considered as a
compromise state between crystallinity and amorphousness in
which later is originated by the oxygen functional groups. The
SEM images as depicted in Figure 2. (a and b) reveal sheets
like morphology of MoS2 NS. For CDs small aggregations of
particles were observed (Figure 2. (c and d)). However, SEM
images are found incompetent to provide pin point
morphological attributes of CDs due to their quantum
dimensional size (<5nm). Henceforth, TEM analysis was
adopted. Low resolution as well as high resolution TEM images
confirmed spherical morphology of CDs (Figure 3). The size
distribution was restrained within a dimensional range of 2.5-
Results and discussion
Materials preparation and characterization
Highly luminescent CDs were obtained by simple one-step and
one-pot hydrothermal synthesis by using soluble starch and
citric acid as carbohydrate precursor. Under the hydrothermal
condition, extensive pyrolysis of hydrocarbons took place
followed by carbonization of the degraded products, which
ultimately gives birth to multifunctional graphitic nano CDs. On
the other hand, MoS2 NS were prepared by exfoliation of
layers form bulk MoS2 by using acoustic power of sonic energy
in an aqueous medium. High energy ultrasound waves peel off
the successive layers from the bulk sample and gives the 2D
nano-sheets. The purified (centrifuged) dispersion of MoS2 NS
was found stable and can be stored on bench-top for several
weeks. Prepared nanostructures were characterized by using
various analytical and spectroscopic tools. UV-vis spectrum of
Figure 1. (a) UV-vis spectrum CDs; (b) UV-vis spectrum of MoS2 and (b) XRD patterns of CDs and MoS2 NS.
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Figure 2. SEM images of (a) and (b) MoS2 NS; (c) and (d) CDs.
5.5 nm (Figure S2). The largest fraction of CDs has a dimension
of 3.5-4.5 nm. The HR-TEM images (Inverse Fast Fourier image
(IFFT) of selected region) showed an interlayer spacing of 0.34
nm. This can be ascribed to slightly exfoliated (001) plane of
graphitic carbon. On the other hand, MoS2 NS seems to
possess 2D sheets like structure. The low resolution TEM
images give evidence for the formation of exfoliated MoS2 NS
(Figure 4. a, b, c and d). These images further showed that
exfoliated MoS2 NS possess the lateral dimension of 200-300
nm. HR-TEM image and IFFT image (Figure 4.e and f) reveal the
periodic arrangement of the atoms on MoS2 NS. The inter-
planar distance was measured as 0.27 nm attributed to (100)
facet of MoS2. The SAED pattern of CDs displayed concentric
cycles indicating the poorly crystalline graphitic phase (inset to
Figure 4.f). For MoS2 NS phase, concentric rings with bright
spots were observed which have been assigned as (100), (103),
(006) facet of MoS2. The TEM results are implicative of XRD
analysis. The EDS analysis and elemental mapping showed the
composition of CDs and MoS2 NS. CDs composed of 32.42% of
O and 67.58% of C while MoS2 NS contains 37.49% of Mo and
62.51% of S (Figure S3 and S4). Elemental analysis verified high
C content in CDs and the overall phase purity of MoS2 NS.
Thus, the overall analysis confirmed successful generation of
CDs and MoS2 nanostructures.
Figure 3. (a) and (b) Low resolution TEM images of CDs, (c) High resolution image of
CDs, (d) IFFT of CDs.
of upconversion emission is not certain. However, it has been
proposed that abnormal photoluminescence originates
through a multi-photon activation process [32]. Simultaneous
absorption of two or more photons leads to the emission of
light at a shorter wavelength than the excitation wavelength.
The upconversion luminescence spectra of anti-cTnT-f-CDs
were recorded in the excitation wavelength range of 900-950
nm. An excitation wavelength dependent upconversion
emission was perceived in the green region. Typical excitation-
emission spectra as well as intensity mapping showed a strong
fluorescence at the wavelength region of 920-940 nm with
emission near 530 nm (Figure 5.a and d). In dynamic energy
transfer processes, position of emission wavelength of donor
and absorption wavelength of acceptor is vital specifically to
constitute an effective FRET process. Here, CDs/MoS2 NS
couple seems to be effective by virtue of the overlapping
absorption-emission spectral regions. Absolute quantum yield
(Ф) value was measured at the wavelength of 940 nm by
following an integrated sphere model, which was found as
2.98%. Similarly, the CIE upconversion coordinates show
indices of (x,y) (0.273, 0.254). This gives evidence about a
green emission. Thus, overall results showed good optical
properties of CDs which can be utilized to design fluorescence
based sensing platforms.
Optical properties
One of the most relevant optical properties of CDs is
wavelength dependent fluorescence. CDs possess typical
optical absorption in the UV-vis region and emission in the
red/green region. The excitation wavelength dependent
emission spectra of anti-cTnT-f-CDs are shown in Figure S5.
Intensity mapping showed a strong fluorescence in the
wavelength range of 320-370 nm. Absolute quantum yield (Ф)
was calculated as 7.45% at the excitation wavelength of 360
nm with chromaticity index of (x,y : 0.151,0.123). These indices
indicate a strong emission in the blue region. In addition to
conventional down conversion fluorescence, CDs also exhibit
NIR upconversion photoluminescence. The precise mechanism
Study of FRET between anti-cTnT-f-CDs/MoS2 NS
Generally, 2D nanosheets have been reportedly used as
quencher in different sensing platforms. MoS2 NS can absorb
emissive radiation over a wide range of wavelength as well as
possess the ability to interact specifically with certain bio-
entities. Such characteristics make them suitable to act as
quencher. Exfoliated layers of MoS2 can be considered as S-
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Figure 4. (a), (b), (c) and (d) Low resolution TEM images of exfoliated MoS2 NS; (e) high resolution TEM image of MoS2 showing atomic arrangement; (f) IFFT of MoS2 (inset SAED
pattern).
Figure 5. Excitation-emission spectra of (a) anti-cTnT-f-CDs, (b) anti-cTnT-f-CDs/MoS2 NS (50 µM) and (c) anti-cTnT-f-CDs/MoS2 NS/cTnT; intensity mapping of (d) anti-cTnT-f-CDs,
(e) anti-cTnT-f-CDs/MoS2 NS and (f) anti-cTnT-f-CDs/MoS2 NS/cTnT.
Mo-S sandwich structure in which each Mo is coordinated in a of MoS2 NS. Intensity quenching was monitored in the whole
trigonal prismatic geometry to six S atoms [33, 34]. With such region of 900-950 nm as shown in Figure 5.b and f. Time-
unique morphological characteristic, MoS2 NS can absorb resolved fluorescence measurements are also carried out with
quantum dots via van der Waals force between nucleobases variation of MoS2 NS concentration. The life time
and the basal plane of MoS2. Additionally, a strong interaction measurements depicted in Figure 7 showed a clear decrease in
exists between CDs and MoS2 NS by means of delocalized π fluorescence life time after the addition of MoS2 NS. The
electrons and multi-functional groups. Such interactions are average decay time of anti-cTnT-f-CDs and anti-cTnT-f-
momentous to bring both the nanostructures to their close CDs/MoS2 NS (50 µM) were found as 8.2 and 4.1 ns,
proximity. Therefore, a combination of CDs/MoS2 NS is respectively. The time-resolve spectra were fitted according to
expected noteworthy to establish an effective excited state the following exponential equation.
energy transfer dynamics between them. Prior to sensing
ꢎ
ꢋꢂꢉ
ꢌꢉ
application, a details study on quenching efficiency of the
nano-couple was under taken. A varying concentration of
MoS2 NS (5-50 µM) was used to bring turn off fluorescence of
anti-cTnT-f-CDs. As depicted by Figure 6.a, fluorescence
intensity decreased proportionally with the increase in
concentration of MoS2 NS. An eight fold decrease in
fluorescence intensity was observed on the addition of 50 µM
ꢁ ꢃ
ꢀ ꢂ ꢄ ꢅ ꢆ ꢇ ꢈꢉ exp ꢊ
ꢍ
ꢉ
where n is the number of discrete emissive species, A is a
baseline correction, Bi is the pre-exponential factor and τi is
the excited-state fluorescence lifetime associated with the ith
component. The fitted decay profile of anti-cTnT-f-CDs was
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Figure 6. (a) Quenching of fluorescence intensity of anti-cTnT-f-CDs by MoS2; (b) Relation between relative change in fluorescence intensity of anti-cTnT-f-CDs and MoS2
concentration (non-linear upward Stern-Volmer curve).
found to follow 3rd order exponential. The decay times have
In order to understand the quenching mechanism, we
been measured as τ1=8.35 ps (16.68%), τ2=1.99 ns (21.69%), plotted relative change in intensity (F0/F) against the
and τ3=8.35 ns (61.63%) for anti-cTnT-f-CDs. The multi concentration of MoS2 NS. Figure 6.b depicts a non-regular and
component decay profile can be ascribed to the existence of non-linear pattern of change in intensity with quencher
multiple radiative states within carbon dot nano structure. concentration. This is quite a contradictory type of pattern to
While precise identification of these radiative species is a big advocate a dynamic (collisional) quenching process where a
challenge, since the mechanism of fluorescence of CDs is not linear response is the ideal. On the other hand, the time
certainly answered yet. However, surface state transitions are control fluorescence measurements showed variation in
believed to be responsible for such optical behaviour. The lifetime (τ) on the addition of quencher. Generally, static
surface states of carbon nanoparticles contain various quenching occurs with an increase of F0/F, but without
functional groups like carbonyl, carboxyl, epoxy, hydroxyl etc. affecting the lifetime. In case of pure dynamic quenching, the
Each surface state is associated with definite energy levels. All condition of F0/F = τ0/τ is imperative, where the slopes of the
these surface states are not passivated. Due to the variety of linear plots of F0/F vs [MoS2] and τ0/τ vs [MoS2] coincides [37].
possibilities some of them are non-radiative states, which are In the present case we observed F0/F vs [MoS2] and τ0/τ vs
known as traps. When more than one passivated surface state [MoS2] curves with divergent slopes. The most significant thing
responded to the excitation energy, multiple decay observed in the quenching process is a clear reduction in the
components are observed [35, 36]. The decay times are found fluorescence lifetime on the addition of MoS2 NS with
to be decreased when MoS2 is added into anti-cTnT-f-CD consequent decrease in the fluorescence intensity. Therefore,
solution. On addition of 50 µM of MoS2 NS, the decay times τ1, we can assume a combined static and dynamic quenching
τ2, and τ3 are decreased from 8.35 ps, 1.99 ns, 8.35 ns to 6.28 process between anti-cTnT-f-CDs and MoS2 NS which is
ps, 1.02, 5.35 ns, respectively.
effective to bring quenching of the fluorescence. Such unusual
process can be expressed in terms of modified Stern-Volmer
equation as follow [38].
ꢏ
ꢐꢑ
ꢁ
ꢔ
ꢖꢃꢁ ꢖꢃ
ꢔ
ꢄ 1 ꢆ ꢒꢓ MoSꢕ 1 ꢆ ꢒꢗ MoSꢕ
ꢏ
where F0 and
F denote the steady-state fluorescence
intensities in the absence and in presence of MoS2 NS, KS
represents static quenching constant and KD represents
dynamic quenching constant. The quadratic term [MoS2]2 in
the modified Stern-Volmer equation deviates the plot of F0/F
vs [MoS2] from linearity. Thus, the nonlinear upward Stern-
Volmer plot suggests that quenching might be contribution of
a combined dynamic and static process. In general static
process originates through the complexation in the ground
state before excitation. For the anti-cTnT-f-CDs/MoS2 NS nano-
couple such complexation might be operative courtesy to the
functional groups present in CDs.
Figure 7. Time resolve decay profiles of anti-cTnT-f-CDs, anti-cTnT-f-CDs/MoS2 NS and
anti-cTnT-f-CDs/MoS2 NS/cTnT.
To understand the energy transfer dynamics between anti-
cTnT-f-CDs and MoS2 NS we can follow a non-radiative energy
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transfer mechanism viz. Förster resonance energy transfer excited state of CDs, which leads to quenching of emission
(FRET). For FRET to occur, overlapping of absorption spectrum intensity [39].
of acceptor with emission spectrum of donor is required. Analytical performance of anti-cTnT-f-CDs /MoS2
Generally, combined energy transfer efficiency (ϕ) of FRET can
The mechanism of cTnT immunosensing relies on the distance
be expressed according to the following agreement
dependent FRET between anti-cTnT-f-CDs and MoS2 NS. The
schematic presentation of the working principle is depicted in
Scheme 1. As discussed in the above sections, anti-cTnT-f-CDs
1
ꢘ ꢄ
ꢎ
ꢚ
1 ꢆ ꢙ
ꢛ
ꢚꢐ
absorb NIR radiation and emit green radiation (530 nm).
However, in presence of MoS2 NS (50 µM), upconversion
fluorescence gets quenched. Such situation arises only when
anti-cTnT-f-CDs/MoS2 pair takes positions in close proximity to
each other. Any entity capable of making separation between
them would interfere the energy flow process and thus restore
the quenched upconversion intensity. Cardiac cTnT is a bulky
37 ku protein molecule and possesses the capability to push
steric disturbance between anti-cTnT-f-CDs/MoS2 FRET couple.
In presence of cTnT, anti-cTnT-f-CDs bind them very strongly
courtesy to specific antibody/antigen interaction (cTnT/anti-
cTnT). These prominent interactions causes the detachments
of quantum sized CDs from the 2D MoS2 NS. As a result,
fluorescence is recovered with change in intensity (F-F0)
proportional to the concentration of cTnT. In the whole
process, the role of anti-cTnT is vital. It brings cTnT to the close
proximity of CDs exclusively out of all other bio-entities that
may present in a real sample and provides the requisite
stability to interfere between the donor/acceptor couple.
To endorse the potential application of anti-cTnT-f-
CDs/MoS2 toward cTnT sensing, static and dynamic
fluorescence measurements were carried out in presence of
varying concentration of cTnT. A fixed anti-cTnT-f-CDs/MoS2
was taken as florescence probe and static florescence
response was recorded in presence of cTnT in the
concentration range of 0.1-100 ng mL-1. The pH of the medium
was maintained at 7.4. Here it is pertinent to mention that
fluorescence intensity of CDs vastly depends on the pH of the
medium. We found that CDs possess a stable emission in the
mid pH range showing maximum fluorescence intensity. In
where r is the distance between donors and acceptors, and r0
is the fluorescent distance of donors and acceptors when the
transfer efficiency is 50% and n=6. The energy transfer
efficiency is inversely proportional to the 6th power of the
distance between the donor and acceptor. The time resolved
fluorescence measurements confirmed the existence of non-
radiative flow dynamics between anti-cTnT-f-CDs and MoS2.
The decay profiles as well as their corresponding steady state
fluorescence spectra as depicted in Figure 6.a and Figure 7
show gradual decrease in fluorescence lifetime as well as
intensity. Such behaviour is indicative of an effective energy
transfer between the donor and acceptor. Therefore, we can
introduce the rate of resonance energy transfer (k) between
anti-cTnT-f-CDs and MoS2 NS according to the following
equation.
1
1
ꢜ ꢄ
ꢋ
ꢌꢁanti ꢋ cTnT ꢋ f ꢋ CDs/MoSꢕꢃ ꢌꢁanti ꢋ cTnT ꢋ ꢝ ꢋ CDsꢃ
The calculated k value is found to be 1.2 × 108 s−1. As reported
in the previous sections, anti-cTnT-f-CDs possess upconversion
emission in the green region, while MoS2 NS have a strong
absorption in the 500-700 nm range. Hence, this can bring an
effective spectral overlap of donor/acceptor couple which in
turn apparently initiate a resonance type energy transfer
dynamics. Further, CDs can transport an excited electron-hole
pair to acceptor MoS2 NS. Such charge transfer process
provides a mechanism for non-radiative deactivation of the
Scheme 1. Detection of cTnT based on FRET dynamics between anti-cTnT-f-CDs and MoS2 nano-couple.
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Figure 8. (a) Restored fluorescence intensity of anti-cTnT-f-CDs/MoS2 in presence of cTnT; (b) Calibration curve for estimation of cTnT.
highly acidic and basic condition fluorescence intensity is accuracy level of 101.4±3.7. Regression analysis showed a co-
affected (Figure S6.a). Therefore, pH of 7.4 was maintained to relation co-efficient of 0.99. The value of LoD and LoQ have
achieve highly intense emission as well as to mimic been determined and found as 0.12 and 0.38 ng mL-1. We
physiological pH condition. Fluorescence spectra as shown in compared theses values with different other research based
Figure 8.a reveal a gradual increase in fluorescence intensity techniques. Table S1 shows that the values obtained for the
with increase in cTnT concentration in buffer as the medium. current assay are well comparable or even lower than the
Such enhancement of intensity was observed for the entire reported ones. However, compared to commercial assays, the
frequency region of 900-950 nm (Figure 5.c and f). We have measured LoD value is relatively higher. Table S1 also highlight
also verified restoration of fluorescence at various levels of pH, performance characteristics of some of the commercially
which confirmed maximum enhancement in intensity in the available cTnT assays. For commercial cTnT assays, LoD varies
mid pH region (Figure S6.b). Similarly, the effect of incubation between the range of 0.01 and 0.16 ng mL−1. In this context,
time was further optimized. Stable restored fluorescence we would like to point out few important facts. While
intensity was observed after 12 min of incubation (Figure S6.c). reporting LoD, we should remember that the precise threshold
Therefore, we followed a 15 min of incubation time for the amount of cTnT in cardiac patient in not certain. That is why,
current cTnT detection method. Dynamic fluorescence the description of cTnT amount in an analytical assay
measurements further confirmed FRET controlled detection of advocates the concept of 99th percentile concentration
cTnT. The decay profile of anti-cTnT-f-CD/MoS2 showed reflecting the limiting concentration below which 99% of a
increase in fluorescence life time in presence of cTnT. The healthy reference population will fall [40]. 99th percentile
original decay times of anti-cTnT-f-CDs/MoS2 i.e. τ1=6.28 ps, concentration is not absolute and depends on the sensing
τ2=1.02 ns, and τ3=5.35 ns encountered increasing values with technique and sensing platform. Different assays and different
τ1=8.35 ps, τ2=1.32 ns, and τ3=8.07 ns in presence of 50 ng mL-1 labs use different cut off values of 99th percentile
of cTnT (Figure 7). Such tendency to recover original life time concentration for diagnosis. State of the art shows that this
confirmed a significance hindrance in the flow of resonance concentration may vary between 0.01 and 0.2 ng mL-1 [41]. For
energy from anti-cTnT-f-CDs to MoS2 NS in presence of cTnT. commercial assays, LoD values are evocative only with
Such results provide evidence for an effective energy transfer reference to an internally calibrated 99th percentile
control in the cTnT detection process.
concentration. Therefore, significance of apparently higher
In order to evaluate the aptness of the current cTnT assay, a LoD values of research based methods compared to the
linear curve fitting procedure was adopted establishing the commercial methods cannot be ignored and proper
relationship between enhanced fluorescence intensity and justification is possible only after the detail study of 99th
concentration of cTnT. An excellent linear response was percentile concentration. Thus, the obtained results are
perceived in the concentration range of 0.1-50 ng mL-1. conclusive and noteworthy to highlight the potentiality of anti-
However, above cTnT concentration of 50 ng mL-1, deviation of cTnT-f-CDs/MoS2 as sensing platform for detection of cTnT.
linearity was observed. The concentration of cTnT (in the range
Detection of cTnT was carried out in human blood serum
of 0.1-50 ng mL-1) and the corresponding change in intensity also. The basic idea was to replicate real sample analysis and
can be described by the linear agreement (F- to confirm presence of any potential inference from the other
F0)=26.894[cTnT]+65.532 (Figure 8.b). The significance of the bio-entities that may be present. Similar to buffer, an
whole analytical procedure was validated by statistical analysis enhanced fluorescence intensity was recorded and the
(Supporting Information). The above equation was found restored fluorescence intensity was co-related to the cTnT
significant with a recovery percentage of 101.4% with an concentration which shows
a
linear increment. The
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relationship between enhanced intensity and cTnT gratitude towards Director, CSIR-NEIST, Jorhat for his interest
concentration can be expressed by the equation (F- in this work.
F0)=21.538[cTnT]+58.223. The LoD and LoQ value for the
detection of cTnT in serum was found as 0.99 and 3.01 ng mL-1.
Notes
These values are slightly higher than the values calculated for
cTnT detection in buffer. Such type of deviation may occur by
ORCID
non-specific absorption originated from the other
Satyabrat Gogoi: 0000-0002-8860-5615
biomolecules present in blood serum. Thus, shows a minor
Raju Khan: 0000-0002- 3007-0232
influence of the intefrents on the sensing performance of anti-
cTnT-f-CD/MoS2 sensing probe for detection of cardiac cTnT in
a replica of real sample. Generally, blood serum contains other
proteins and bio-entities abundantly at
a much higher
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results are good enough to highlight the analytical
performance of anti-cTnT-f-CDs/MoS2 in the detection of cTnT.
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