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Rapid Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes Mojtaba Shamsipur, Fatemeh Molaabasi, Saman Hosseinkhani, and Fereshteh Rahmati Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03824 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Analytical Chemistry

Rapid Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes Mojtaba Shamsipur,*,† Fatemeh Molaabasi,‡ Saman Hosseinkhani,§ and Fereshteh Rahmati§ † ‡ §

Department of Chemistry, Razi University, Kermanshah, Iran Department of Chemistry, Tarbiat Modares University, Tehran, Iran Department of Biology, Tarbiat Modares University, Tehran, Iran

ABSTRACT: Cytochrome c (Cyt c) is an important biomarker in cell lysates for early stage of apoptosis or anti-cancer agents. Here, two novel label-free fluorescence assays based on hemoglobin-stabilized gold nanoclusters (Hb/AuNCs) and aptamer-stabilized silver nanoclusters (DNA/AgNCs) for analysis of Cyt c are presented. The heme group of the protein induces sensitive sensing platforms accompanied by the decreased fluorescence of both metal nanoclusters. The quenching processes observed found to be based on the fluorescence resonance energy transfer mechanism from Hb/AuNCs to Cyt c and photoinduced electron transfer from DNA/AgNCs to the aptamer-Cyt c complex. The linear range for Cyt c was found to be 0-10 µM for Hb/AuNCs and from 0-1µM for DNA/AgNCs, with limits of detection of ~15 nM. Based on strong binding affinity of DNA aptamers for their target proteins, the DNA/AgNCs probe was successfully applied to the quantitative determination of Cyt c in cell lysates, which opens a new avenue to early diagnostics and drug screening with high sensitivity. Compared to the conventional Western blot method, the presented assays are low cost, easy to prepare the fluorescent probes and sensitive, while overall time for the detection and quantitation of Cyt c from isolated mitochondria is only 20 min. The proposed method for Cyt c detection may also be useful for the study of those materials that cause mitochondrial dysfunction and apoptotic cell death.

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INTRODUCTION Apoptosis, the programmed cell death, is an active regulatory response in both physiological and pathological conditions. Signaling for this important process is occurred through some multiple pathways, initiated by triggering events either within the cell via DNA damage and/or cytoskeleton disruption or from outside the cell, based on ligation of the death receptors.1,2 The intrinsic apoptotic pathway is consisted of the permeabilizination of the outer mitochondrial membrane and the release of cytochrome c (Cyt c), which can be used as an indicator for the apoptotic process in the cell.1,3-6 For this reason, the measurement of Cyt c, as an effective bio-marker, in cell lyates can lead to a better understanding of certain diseases on a cellular level.7,8 It is well known that cytochrome c plays a critical role in the process of electron transfer in oxidative phosphorylation via the redox reaction of its iron content.7,9 Apoptosis, the programmed cell death, is an active regulatory response in both physiological and pathological conditions. Signaling for apoptosis occurs through multiple pathways that are initiated by triggering events either within the cell via DNA damage and/or cytoskeleton disruption or from outside the cell by ligation of death receptors.1,2 The intrinsic apoptotic pathway involves the permeabilizination of the outer mitochondrial membrane and the release of Cyt c, which is used as an indicator for the apoptotic process in the cell.1,3-6 For this reason, measurement of Cyt c, as an effective bio-marker, in cell lyates can lead to better understanding of certain diseases on a cellular level.7,8 Cytochrome c plays an essential role in the process of electron transfer in oxidative phosphorylation by the redox reaction of its iron atom.7,9 A variety of experimental methods have already been developed for the detection of Cyt c. These include fluorometry,8 spectrophotometry,10 mass spectrometry,11 chemiluminescence,12 electrophoresis,13 HPLC14 and electrochemistry,15,16 Very recently, an aptameric nanosensor for fluorescence activation imaging of Cyt c released from mitochondria has also been developed.17 The proposed methods do not simultaneously possess the advantages of simplicity, rapidity, selectivity, and simplicity;1,16 i.e., although most of these methods possess high sensitivity, they are multi-step and time consuming processes (Table 1).1,11,15,16,18 Moreover, the instrument-based methods (e.g. HPLC and ICP/MS) in spite of rapid analysis, are expensive, complicated, and also require extensive pretreatment of the sample.15,16 The reported electrochemical biosensors suffer 2 ACS Paragon Plus Environment

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from lack of selectivity for the determination of Cyt c, due to the interaction of other small proteins with the recognition elements viz.1,15 Conventionally, Cyt c release studies are carried out via the use of Western blotting and the enzyme-linked immunosorbent assay (ELISA), which impose a number of limitations.3,9,13-15 Apart from being tedious, troublesome, lengthy and inconvenient for the processing of multiple samples, the Western blot analysis is an inherently semi-quantitative method in apoptotic cells and may be complicated by the cross-reactivity of the anti-Cyt c that binds to some other cellular proteins.9,10,14,16,19 Also, the signal amplification in ELISA mediated by an enzymatic reaction is limited by the availability of substrates.19 The fluorescent metal nanoclusters (NCs) are new class of materials, exhibiting a molecular-like spectroscopic behavior.17,20 Gold and silver nanoclusters have received considerable attention in the detection of a wide variety of analytes.21,22 Due to their excellent fluorescence properties and suitability for applications in the fields of chemical-sensing,18 in vivo biological imaging,23 and in vitro bioassays,24 proteins as green-chemical templates are highly attractive for the synthesis, characterization, and applications of gold nanoclusters, This is due to the fact that the amine, carboxyl, and thiol groups present in proteins can serve as both reducing and stabilizing agents in such formulations.25-27 Recently, the use of oligonucleotides (known as aptamers) with high binding capabilities has received an increasing attention. Compared with antibody-antigen reaction-based immunoassays, such aptamer-based biosensors (aptasensors) have the advantages of easy preparation, stability, reusability, and general availability for almost any given protein. 28,29 Thus, the aptamer/AgNCs assembly has widely served as both a fluorescent label and a specific binding ligand.30,31 Herein, we introduce the development and use of two fluorescent recognition systems, namely, a hemoglobin-templated AuNCs (Hb/AuNCs) with blue emission26,27 and an aptamer-templated AgNCs (DNA/AgNCs) with red emission,32,33 for selective and sensitive detection of Cyt c. The quenching processes are observed based on the fluorescence resonance energy transfer (FRET) from Hb/AuNCs to Cyt c and the photoinduced electron transfer (PET) from DNA/AgNCs to the aptamer-Cyt c complex. Both nano-biosensors offered the potential for simple, low-cost, and rapid tracking of Cyt c in the culture medium released from isolated mitochondria or from permeabilized cells when apoptosis was occurred with CCLR buffer, and for anti-cancer drug screening based on the assessment of the amount of Cyt c released from the apoptotic cells after the exposure to doxorubicin as anti-cancer drug. To the best of our 3 ACS Paragon Plus Environment


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knowledge, there is no previous report addressing the ability to measure selective apoptotic biomarker protein using nanocluster-based sensing systems in the cell culture.

EXPERIMENTAL SECTION Chemicals and Materials. Oligonucleotide sequences Cyt c-1: 5′-CCT-CCT-TCC-TCCAGT-GTG-AAA-TAT-CTA-AAC-TAA-ATG-TGG-AGG-GTG-GGA-CGG-GAA-GAA-GTTTAT-TTT-TCA-CACT-3′ and Cyt c-2: 5′-CCT-CCT-TCC-TCC-CGT-GTC-TGG-GGC-CGACCG-GCG-CAT-TGG-GTA-CGT-TGT-TGC-3′ were obtained from Faza Biotech Co. (Tehran, Iran). Chloroauric acid was obtained from Alfa Aesar (Ward Hill, MA, UK). Cyt c, human serum albomin (HSA), bovine serum albomin (BSA), lysozyme (Lys), Insulin, adenosine triphosphate (ATP), glutathione (GSH) and Immunoglobulin G (Rabbit IgG) were obtained from SigmaAldrich (St. Louis, MO) and used without further purification. Hemin, as an iron-containing porphyrin, was purchased from Sangon Biotechnology (Shanghai, China). NaOH, AgNO3, NaBH4, Na2HPO4, NaH2PO4 and NaCl salts were purchased from Merck (Darmstadt, Germany). All stock solutions were prepared using deionized water with a resistivity not less than 18 MΩ cm (Milli-Q, Bedford, MA). Other chemicals involved in this work were of analytical grade and used as received. Phosphate buffer solution (PBS, 0.20 M, pH 7.4) was prepared by mixing a stock standard solution of Na2HPO4 and NaH2PO4 and adjusting the pH with NaOH. The experimental procedures and protocols for preparation of hemoglobin were done according to the method of Williams and Tsay.34 The concentration of the protein was determined by the method of Antonini and Brunori (ε415nm = 125 mM-1 cm-1 or ε541nm = 13.8 mM-1 cm-1 per heme). The concentrations were ≥100 µM to avoid the formation of a considerable amount of dimers, which should always be ≤5%.35

Apparatus. Spectrofluorimetric measurements were performed using a Perkin-Elmer LS50B instrument (Perkin-Elmer, U.K.). The emission spectra for Hb/AuNCs and DNA/AgNCs, were recorded over the wavelength ranges of 390-600 nm and 590-770 nm upon excitation at 365 nm and 560 nm, respectively, at 1500 nm min-1 scan rate. The spectral band pass was set at 15 and 20 nm for excitation and emission, respectively. The absorption spectra were recorded on


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a Model Scinco UV S-2100 (Cinco, Korea). The TEM characterization was performed on a Tecnai G2 F20 (Philips, Holland) with an accelerating voltage of 150 kV.

Synthesis of Metal Nanoclusters. The synthesis of Hb/AuNCs probe was mainly based on a protocol described previously.26,36 The fluorescent DNA/AgNCs probes were synthesized using the method developed by Dickson and coworkers.32 In the Supporting Information (SI) are given the detailed synthetic procedures (section SI-1). Assay of Cyt c. 200 μL of the prepared Cyt c-2 stabilized AgNCs were titrated by successive additions of a given concentration of Cyt c standard solution and incubated for 20 min at 27 ºC after each addition. At an excitation wavelength of 560 nm, the fluorescence spectra of the DNA/AgNCs were recorded over the wavelength interval of 590-770 nm, a linear correlation between the fluorescence intensity and Cyt c concentration was established at the maximum emission wavelength of 610 nm.

Cell Culture and Apoptosis Induction. Detailed procedure for the culture and apoptosis induction of embryonic kidney cells (HEK293T) is given in SI section (SI-4).37

Detection of Released Cytochrome c. Quantitative analysis of the Cyt c in the culture medium released from apoptotic cells was performed by standard addition method to eliminate any matrix effect. Consequently, aliquots (1.0 μL) of this culture medium containing lysate from 1.0 × 105 cells were spiked with a 50 μM standard Cyt c solution (from 0.75 to 0.3 µM) in DNA/AgNCs solution. For every addition, the mixture solution was left to react at room temperature (27 °C) for 20 min and then the fluorescence intensities were measured at an excitation wavelength of 560 nm over an emission wavelengths interval of 590-770 nm. The widths of the excitation and emission slits were set to 15.0 and 20.0 nm, respectively, at a scanning speed of 1500 nm min-1.

RESULTS AND DISCUSSION



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Cyt c-Induced PL Quenching of Hb/AuNCs. Preliminary experiments revealed that the addition of Cyt c to the blue Hb/AuNCs fluorescent probe results in strong quenching of its fluorescence intensity. Figure 1A shows the UV-vis spectrum of Cyt c (a) and fluorescence emission spectrum of Hb/AuNCs in 10 mM PBS of pH 7.4 upon excitation at 360 nm (b). As seen, Cyt c has an absorption maximum at 410 nm due to the heme group of the Cyt c protein,7 whereas Hb/AuNCs shows a photoluminescence (PL) emission maximum at 440 nm. As seen from Figure 1A, there is a significant spectral overlap between the emission spectrum of Hb/AuNCs and the absorption spectrum of Cyt c, indicating the feasibility of a FRET from Hb/AuNCs probe to Cyt c.38 Thus, in the next step, the region of integral overlap was used to calculate the rate constant for the FRET system, kF, and the critical energy transfer distance, R0 (a distance at which the transfer efficiency is 50%), between Hb/AuNCs (as donor) and Cyt c (as acceptor) using the Förster relation: 26,39,40 kF = (1/τD) (R0/r)6

with R0 = 0.211 (k2 n-4 υD J(λ))1/6 (in Å)

Here, τD is the donor radiative lifetime, n is the refractive index of the medium, k2 is the orientation factor, υ is the quantum yield of the donor, and J(λ) is the spectral overlap integral. In this case k2 = 2/3, n = 1.33, φ = 0.028, and J(λ) = 3.82 × 1016 M-1 cm-1(nm), as approximated from the following equation:26,39,40

J(λ ) = ∫

(λ) ε(λ)λ

∫

λ (λ) λ

where F(λ) and ε(λ) represent the fluorescence intensity of the donor and the molar extinction coefficient of the acceptor, respectively. It should be noted that the quantum yield υ of AuNCs was determined by a comparative method, using quinine sulfate in 0.1 M H2SO4 with known quantum yield of υ = 0.54 as a reference fluorophore (see section SI-3 for more detail).40 Consequently, based on this information, the R0 value was calculated and as 52.5 Å, much less than the critical R0 value ~100 Å, indicating an efficient energy transfer between the donoracceptor pair.39 A schematic representations of FRET from Hb/AuNCs to Cyt c is given in Figure 1B. 6 ACS Paragon Plus Environment

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(Figure 1)

To further support the mechanism of sensing system, its tendency at pH 7.4 towards the heme containing compounds Cyt c, hemin and Hb (pI=6.8) and a series of other species including BSA (pI=4.7), HSA (pI=4.7), Lys (pI=11.1), Insulin (pI=5.3), IgG (pI=6.4-9.0), ATP, and GSH was studied and the results are shown in Figure 2A. As seen, the strongest quenching in the fluorescence of probe was observed for hemin, Hb, and Cyt c, all containing the heme group, while the presence of other proteins (i.e., BSA, HSA, Lys, Insulin, and IgG) as well as ATP and GSH led to slight changes in the PL of Hb/AuNCs probe, indicating that the decrease in fluorescence intensity is induced by energy transfer from the Hb/AuNCs donor probe to the heme acceptor group. It should be noted that, based on our previous experimental results26,27 and other literature reports,33-35 the hemoglobin (Hb) molecules surrounding the surface of Hb/AuNCs probe would not quench the fluorescence of the AuNCs and, thus, does not interfere with the detection of Cyt c, due to the hydrophobic microenvironment changes of the heme group in Hb (Figure S3 in SI).26 In addition, it has been reported that AuNCs show an increase in reductive ability with decreasing their core size,22,41 in comparison with gold nanoparticles, so that one cannot rule out about the possibility of heme-induced PL quenching of the Hb/AuNCs through the electron transfer from the nanocluster to the heme group. As also indicated in Figure 2B, maximum quenching occurred in less than 5.0 min incubation by adding 10 µM Cyt c. Meanwhile, the FRET process was found to enhance by increasing concentration of Cyt c and, consequently, resulting in decreased photoluminescence of the Hb/AuNCs at 440 nm, at an excitation wavelength of 360 nm (Figure 2C). To explore the feasibility of using this approach for quantitative analysis, the corresponding fluorescence quenching data were used to construct a Stern-Volmer plot of F0/F vs. Cyt c concentration, based on F0/F = 1 + KSV [Cyt c], and the resulting plot is shown in Figure 2D. As seen, the Stern-Volmer plot showed good linearity from 0 to 10 μM of Cyt c with a correlation coefficient of R2 = 0.998 and a KSV value of 9.6 × 104 M-1. The resulting detection limit (DL), based on a signal-to-noise ratio of 3, was 14.3 µM. The DL thus obtained is lower than or comparable to those of most fluorescent and electrochemical sensors previously reported for Cyt c (see Table 1).

(Figure 2) and (Table 1) 7 ACS Paragon Plus Environment


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Cyt c-Induced PL Quenching of DNA/AgNCs. The synthetic procedure for the oligonucleotide/AgNCs is given in the Experimental and SI parts (SI-1). The resulting sensing strategy relies on the use of a single oligonucleotide structure, which is competent to work both on the synthesis of AgNCs and on the target bio-recognition. Considering the conformational change in the aptamer after protein binding, we employed two recognition strands Cyt c-1, Cyt c2 (Chemicals section) to associate with the AgNCs-nucleation. The process was directly followed by fluorescence spectroscopy. Meanwhile,

the

influences

of

the

oligonucleotide

concentration

and

the

+

oligonucleotide/Ag mole ratio on the synthesis of the AgNCs were also investigated (Figures S4 and S5 in SI). The results revealed that the silver nanoclusters with the strongest PL emission were

generated

at

Cyt-c1

and

Cyt-c2

concentration

of

100

µM.

The

optimal

DNA/AgNO3/NaBH4 molar ratios were found to be 1:24:24 for Cyt c-1 with a green emission (λex = 470 nm) and 1:18:18 for Cyt c-2 with a red emission (λex = 560 nm) (Figures 3A and 3B). The emission spectra as a function of excitation wavelength for Cyt c-1 and Cyt c-2 capped AgNCs are given in Figure S6. Transmission electron microscopy (TEM) was also utilized to characterize the size of aptamer/ANCs (Figure S7). As seen, most of the AgNCs present possess an average diameter of 3.1 ± 0.4 nm. However, there are also some larger particles with size of about 6-7 nm, which could be formed as a result of the accumulation of nanoclusters during drying on a Cu grid.7 Additionally, the photostability of the fluorescent DNA/AgNCs based on Cyt c-1and Cyt c-2

was studied under ultraviolet irradiation and the results showed that the fluorescence intensity did not change significantly after irradiation for 30 min, while it showed only ~10% change after irradiation for 2 h (Figure S8). In the next step, the influence of concentration of Cyt c as target on the fluorescence spectra of the Cyt c-1and Cyt c-2 stabilized AgNCs were studied and the results are shown in Figure 3C. As seen, the addition of increasing concentration of Cyt c possesses nearly no fluorescence variation for the Cyt c-1/AgNCs, while the FL intensity of the Cyt c-2/AgNCs sharply decreased with increasing target concentration in solution. These results indicated that the properties of nanoclusters and their response patterns to analytes are highly dependent on the base composition of the DNA template used.42 8 ACS Paragon Plus Environment

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(Figure 3)

The addition of Cyt c to a Cyt c-2/AgNCs solution resulted in a pronounced quenching of the emission of the probe. A possible detection mechanism for this label-free turn-off strategy of the probe for Cyt c detection is displayed in Figure 4. As seen, the high affinity of aptamer for the target protein results in the closing of Cyt c to the silver clusters through the formation of an aptamer/Cyt c complex, which results in the quenching of AgNCs. By comparison of the redox state of the aptamer/Cyt c complex and the energy levels of the DNA/AgNCs (Figure 4), a photoexcitation electron transfer mechanism (PET) could be offered for the turn-off pattern according to the following mechanism:7,43 Cyt c-2/AgNCs + h𝑣 Cyt c-2/AgNCs* + Cyt c (III)

Cyt c-2/AgNCs* Cyt c-2/AgNCs + Cyt c (II)

The oxidation potential of purified DNA/AgNCs was 0.11 V vs Ag/AgCl (0.33 V vs NHE) (Figure S9A), and the Cyt c complex revealed a quasi-reversible redox wave at -0.25 V vs Ag/AgCl (-0.028 V vs NHE, Figure S9B), corresponding to the Fe(III)/Fe(II) couple of the heme present in Cyt c. Nevertheless, the oxidation peak of the AgNCs almost disappeared upon addition of Cyt c. On the basis of these potential test results, the excited electrons of the DNA/AgNCs can be transferred to the Cyt c complex (as depicted in Figure 4).44 It should be noted that a similar mechanism has been proposed for G-quadruplex/hemin complex, in the literature.45,46 In addition, it has been reported that the PET effect is highly sensitive to the distance between the DNA/AgNCs and aptamer/Cyt c complex and the structure of DNA matrix.45 Considering the length of the Cyt c-1 aptamer (73 mers length), it can be concluded that the aptamer/Cyt c-1 complex is not sufficiently close to the AgNCs to initiate the photoexcitation electron transfer46 and, consequently, no effective quenching was observed in this case. Meanwhile, in the case of Cyt c-2 (51 mers length)/AgNCs, the absorption of the DNA/AgNCs did not significantly change in the presence of Cyt c, indicating that fluorescence quenching in this system could not occur by the formation of a nonfluorescent ground-state complex.45 Furthermore, the absorption of the Cyt c (Figure 1A, spectrum a) is located at ∼400 nm, which does not overlap with the emission spectrum of both DNA/AgNCs platforms (λem= 530 for Cyt



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c-1 and 620 nm for Cyt c-2) and, thus, eliminating any possibility for a FRET quenching route (Figure S10 in SI).22,45,46

(Figure 4)

Figure 5A shows the time-dependent luminescence changes of DNA/AgNCs upon the addition of 1 µM Cyt c. As seen, maximum quenching occurs in less than 20 min of incubation and remained more or less constant up to 30 min, indicating a relatively slow kinetics of formation of the equilibrium state of the aptamer/Cyt c complex.47 Accordingly, a time interval of 20 min was selected for further studies. To evaluate the specificity of the protein detection assay towards Cyt c, the interfering effects of the addition of equimolar amounts of some other proteins (i.e., Hb, BSA and Lys, Insulin, and IgG) of varied structures as well as hemin, ATP and GSH on Cyt c-2/AgNCs probe was tested and the results are shown in Figure 5B. As is obvious from Figure 5B, and in contrast to the fluorescence quenching observed for Cyt c binding, the PL intensity of the probe for nonspecific proteins does not change significantly during the measurement period (~20 min). However, when 1 µM of hemin and Hb were added to the probe solution, within a few minutes, the PL signals were decreased slightly, implying that the FL quenching can be originated from the PET between AgNCs and the heme group present. It is note mentioning that the heme proteins have their own resources, e.g., Cyt c is found to loosely associate with the inner membrane of the mitochondria, while hemoglobin exists in the red blood cells. It is also well known that, in the cell lysate, no additional hemin or Hb exist in the presence of Cyt c, and vice versa in the presence of Hb or hemin there is not any exogenous Cyt c present.37 Thus, this novel probe possessed excellent selectivity for the diagnosis of early-stage apoptotic cells by detection of Cyt c in cell lysate samples. The proper selectivity observed for Cyt c-2/AgNCs probe towards Cyt c probably results from the specific and appropriate affinity binding of the probe to the target. In order to investigate the ability of Cyt c-2/AgNCs to sensitively detect target protein, different concentrations of Cyt c solutions were tested over a wide range of concentration (0–5 µM, Figure 5C). The results show that the fluorescence intensity decreased with increasing concentration of Cyt c and, finally, the binding saturation between the aptamer and target was occurred at roughly 5 µM. As shown in the inset of Figure 5D, there is a good linear relationship 10 ACS Paragon Plus Environment

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between the fluorescence intensity and Cyt c concentration in the range from 0 to 1.0 µM (y = 0.9354+ 1.8403 C, R2 = 0.9949) with a detection limit of 15.0 nM, based on a signal-to-noise of 3. It is interesting to note that although the linear range and detection limit obtained here are more or less comparable with those reported for most of other reported fluorescence detection methods for Cyt c detection (Table 1), the present method does not require any labeling and/or any signal amplifications. It is notable that the spectrophotometry often suffers from interference. On the other hand, in most biological studies, the reported electrochemical methods by combination with enzymatic activity or antibody are costly and lengthy procedure, while they mainly focused on the mechanism of electron transfer between heme proteins and all kinds of electrodes. Conventional methods for determination of Cyt c release including Western blotting and ELISA-based methods are semi-quantitative, time consuming and tedious.14,37 While, here we introduced a simple, rapid and inexpensive method by using aptamer-stabilized nanocluster assay system.

(Figure 5) Detection of Cyt c release upon induction of apoptosis. Due to its high sensitivity and selectivity, we considered applying the proposed method to the detection of early-stage of apoptosis based on release of cytochrome c from mitochondria. For this study, human embryonic kidney cells HEK293T were treated with CCLR buffer for 24 h to induce the cytochrome c release into the culture medium.37 Within this set, the normal (HEK293T) cells prepared in hypotonic buffer were used as a control cells. To develop a fluorescence assay for Cyt c, in the first step, different volumes (µL amounts) of cell lysates from 100000 cells mL-1 solution were incubated with the nanocluster probes including Cyt c-2/AgNCs (Figure 6) and Hb/AuNCs (Figure S11) followed by fluorescence measurements, similar to those in previous assays. As can be seen from Figure 6A and Figure S8, as the volume of cell lysate in the synthetic nanoclusters solution increases, the resulting fluorescence of DNA/AgNCs is quenched, which is consistent with the higher content of Cyt c in the cell extracts. However, a comparison between Figures 6B and S11A shows that the differences in fluorescence intensity of control cells and the cells released Cyt c in the case of Hb/AuNCs was not as distinct as those obtained it the case of Cyt c-2/AgNCs (Figure 6B). 11 ACS Paragon Plus Environment


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Moreover, the slope of calibration curve from the Cyt c-2/AgNCs was much steeper than that from the Hb/AuNCs. The above results showed that the NIR fluorescent probe gave high specificity to Cyt c at the cellular level because of the specific affinity of aptamer/AgNCs to the protein.28,31,35,47 Additionally, this calibration curve allows us to estimate the average number of cells. Although the Hb/AuNCs sensor cannot be used to distinguish Cyt c in cell lysates, the sensor can be used to estimate the level of heme-containing proteins, i.e., hemoglobin and Cyt c, in biological samples such as serum and blood plasma.32 From the time dependent experiment, carried out using Cyt c-2/AgNCs probe, it was found that the apoptotic cells resulted in significant fluorescent quenching in comparison with the control cells (Figure 6C), which suggesting that the components of the cell lysate such as biothiols do not induce interference with the detection of Cyt c in living cells, reflecting the great potential of the DNA/AgNCs emission probe for further fundamental biology research. As shown in Figure 6C, the cell lines containing larger amounts of Cyt c could induce the formation of more aptamer/AgNCs complex and, consequently, result in significant change in fluorescence intensity with time. 31,45-47

(Figure 6)

Taking advantage of the long excitation wavelength and NIR emission wavelength sensing ability of DNA/AgNCs, in the next step, this probe was applied to the determination of Cyt c in apoptotic cells upon excitation at 560 nm. As the concentration of additional Cyt c increases, the emission intensity at the maximum wavelength of ∼610 nm decreases significantly. The accuracy of the sample analysis was tested by the standard addition method and calculating its recovery. Using the fluorescence intensity originating from 100 cells μL-1, in average, and a kinetic time interval of 20 min (Figure 4A), the concentration of the Cyt c was estimated ∼ 3.8 μM. The results of the recovery tests demonstrated in Table 2 were found to be in the range of 92.0 to 103.4%, which clearly supported the feasibility and reliability of the proposed method. The precision for three replicate detections of Cyt c was also in a satisfactory range of 2.6% to 5.4%. The release of Cyt c upon cell lysis by CCLR has been indicated by western blot37 which is typically used for studies on Cyt c release conducted with isolated


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mitochondria. However, this method is known as a semiquantitative method that cannot give precise information about the amounts and kinetics of release under differing conditions.37 To investigate whether the DNA/AgNCs surviving probe can monitor the level of Cyt c for anti-cancer drug screening in real clinical samples, HEK293T cells were treated with doxorubicin (Table 2), which has been reported useful in treatment of a wide variety of tumors.6,19,48 As it is seen from Table 2, CCLR has released much more Cyt c from the HEK293T (100 cells μL-1 in average) than does doxorubicin. These results confirm that the aptamer coupled with the fluorescent DNA/AgNCs probe can serve as an excellent sensing agent to detect the Cyt c leakage in the culture medium quantitatively as well as after drug treatment without the interference of other substances in the cell.

CONCLUSIONS

In this work, we have reported two different single-step, label-free strategies based on a hemoglobin-stablized AuNCs (Hb/AuNCs) and aptamer-stablized AgNCs (DNA/AgNCs) for selective and sensitive detection of cytochrome c. It was speculated that the mechanism of cytochrome c detection should be different in structurally dissimilar systems. In the case of the well-characterized DNA/AgNCs red emitter, the fluorescence quenching behavior of AgNCs in the presence of cytochrome c found to be primarily due to the efficient PET from DNA/AgNCs to aptamer-Cyt c complex. While a FRET from Hb/AuNCs, as donor, to Cyt c, as acceptor, leads to a dramatic decrease in fluorescence probe intensity. A limit of detection in the nanomolar level (about 15 nM) was achieved for Cyt c detection using both AuNCs and AgNCs based sensing platforms, which allows the extensive dilution of biological fluid samples to minimize the non-specific binding of potentially interfering biomolecules present. In addition, the proposed probes provided a wide range of linear detection of 0 to 10 µM in the case of Hb/AuNCs and from 0 to 1 µM for DNA/AgNCs case, relevant for their clinical applications. Considering the critical importance of the diagnosis of early-stage apoptotic cells, the efficiency of both sensing platforms were evaluated for detection of Cyt c in the apoptotic cells. The aptamer-stablized AgNCs was successfully used as a NIR fluorescent probe for apoptosis monitoring, upon release of Cyt c sample solutions of less than 10 μL. Due to the importance of Cyt c release in early stage detection of apoptosis in rapid drug screening, the developed methods 13 ACS Paragon Plus Environment


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can be used in such screening assays parallel to the intracellular bioluminescence resonance energy transfer assay.

ASSOCIATED CONTENT

Supporting Information Brief statement in nonsentence format listing the contents of the material supplied (in sections SI-1 to SI-13) and Figures S1 to S13, as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *Phone: +98 83 34274515. Fax: +98 83 34274559. E-mail: [email protected]

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the support of this work by Research Council of Tarbiat Modares University and National Elite Foundation of I.R. Iran. The assistance of Ammar Mohseni from Department of Biology of Tarbiat Modares University is also acknowledged. REFERENCES (1) Wen, Q.; Zhang, X.; Cai, J.; Yang, P.-H. Analyst 2014, 139, 2499-2506. (2) Renz, A.; Berdel, W. E.; Kreuter, M.; Belka, C.; Schulze-Osthoff, K.; Los, M. Blood 2001, 98, 1542-1548. (3) Waterhouse, N.; Trapani, J. Cell Death & Differentiation 2003, 10, 853-855. (4) Zhang, S.; Zhu, S.; Yang, L.; Zheng, Y.; Gao, M.; Wang, S.; Zeng, J.-z.; Yan, X. Anal. Chem. 2012, 84, 6421-6428. (5) Song, J. M.; Kasili, P. M.; Griffin, G. D.; Vo-Dinh, T. Anal. Chem.2004, 76, 2591-2594.


Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Torkzadeh-Mahani, M.; Ataei, F.; Nikkhah, M.; Hosseinkhani, S. Biosens. Bioelectron. 2012, 38, 362-368. (7) Cao, M.; Cao, C.; Liu, M.; Wang, P.; Zhu, C. Microchim. Acta 2009, 165, 341-346. (8) Shen, Y.; Yang, X.-F.; Wu, Y.; Li, C. J. Fluores. 2008, 18, 163-168. (9) van Beek-Harmsen, B. J.; van der Laarse, W. J. J. Histochem. Cytochem. 2005, 53, 803807. (10) Appaix, F.; Minatchy, M.-N.; Riva-Lavieille, C.; Olivares, J.; Antonsson, B.; Saks, V. A. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2000, 1457, 175-181. (11) Liu, J.-M.; Yan, X.-P. J. Anal. Atom. Spectrom. 2011, 26, 1191-1197. (12) Li, X.; Liu, H.; He, X.; Song, Z. Appl. Biochem. Biotechnol. 2010, 160, 1065-1073. (13) del Valle-Mondragón, L.; Ramírez-Ortega, M.; Zarco-Olvera, G.; Sánchez-Mendoza, A.; Pastelín-Hernández, G.; Tenorio-López, F. A. Talanta 2008, 74, 478-488. (14) Crouser, E. D.; Gadd, M. E.; Julian, M. W.; Huff, J. E.; Broekemeier, K. M.; Robbins, K. A.; Pfeiffer, D. R. Anal. Biochem. 2003, 317, 67-75. (15) Pandiaraj, M.; Madasamy, T.; Gollavilli, P. N.; Balamurugan, M.; Kotamraju, S.; Rao, V. K.; Bhargava, K.; Karunakaran, C. Bioelectrochem. 2013, 91, 1-7. (16) Luo, D.; Huang, J. Anal. Chem.2009, 81, 2032-2036. (17) Chen,T.-T.; Tian, Tian, X,; Liu, C.-L.; Ge, J.; Chu, X.; Li, Y. J. Am. Chem. Soc. 2015, 137, 982−989. (18) Wu, Z.; Wang, M.; Yang, J.; Zheng, X.; Cai, W.; Meng, G.; Qian, H.; Wang, H.; Jin, R. Small 2012, 8, 2028-2035. (19) Loo, J. F.; Lau, P.; Ho, H.; Kong, S. Talanta 2013, 115, 159-165. (20) Li, J.; Zhu, J.-J.; Xu, K. TrAC Trends Anal. Chem. 2014, 58, 90-98. (21) Qu, F.; Li, N. B.; Luo, H. Q. Anal. Chem. 2012, 84, 10373-10379. (22) Chen, L.-Y.; Huang, C.-C.; Chen, W.-Y.; Lin, H.-J.; Chang, H.-T. Biosens. Bioelectron. 2013, 43, 38-44. (23) Wang, Y.; Chen, J.-T.; Yan, X.-P. Anal. Chem. 2013, 85, 2529-2535. (24) Liu, C. L.; Wu, H. T.; Hsiao, Y. H.; Lai, C. W.; Shih, C. W.; Peng, Y. K.; Tang, K. C.; Chang, H. W.; Chien, Y. C.; Hsiao, J. K. Angew. Chem. Int. Ed. 2011, 50, 7056-7060. (25) Xu, Y.; Sherwood, J.; Qin, Y.; Crowley, D.; Bonizzoni, M.; Bao, Y. Nanoscale 2014, 6, 1515-1524. 15 ACS Paragon Plus Environment


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Page 16 of 29

(26) Shamsipur, M.; Molaabasi, F.; Shanehsaz, M.; Moosavi-Movahedi, A.A. Microchim. Acta 2015, 182, 1131-1141. (27) Molaabasi, F.; Hosseinkhani, S.; Moosavi-Movahedi A. A.; Shamsipur, M. RSC Adv. 2015, 5, 33123–33135. (28) Nam, K.; Eom, K.; Yang, J.; Park, J.; Lee, G.; Jang, K.; Lee, H.; Lee, S. W.; Yoon, D. S.; Lee, C. Y. J. Mater. Chem. 2012, 22, 23348-23356. (29) Song, S.; Wang, L.; Li, J.; Fan, C.; Zhao, J. TrAC Trends Anal. Chem.2008, 27, 108117. (30) Sharma, J.; Yeh, H.-C.; Yoo, H.; Werner, J. H.; Martinez, J. S. Chem. Commun. 2011, 47, 2294-2296. (31) Yin, J.; He, X.; Wang, K.; Xu, F.; Shangguan, J.; He, D.; Shi, H. Anal. Chem. 2013, 85, 12011-12019. (32) Richards, C. I.; Choi, S.; Hsiang, J.-C.; Antoku, Y.; Vosch, T.; Bongiorno, A.; Tzeng, Y.-L.; Dickson, R. M. J. Am. Chem. Soc. 2008, 130, 5038-5039. (33) Li, J.; Zhong, X.; Zhang, H.; Le, X. C.; Zhu, J.-J. Anal. Chem. 2012, 84, 5170-5174. (34) Shao, Q.; Wu, P.; Gu, P.; Xu, X.; Zhang, H.; Cai, C. J. Phys. Chem. B 2011, 115, 8627– 8637. (35) Chilaka, F. C.; Nwamba, C. O.; Moosavi-Movahedi, A. A. Cell Biochem Biophys. 2011, 60, 187-197. (36) Xie, J.; Zheng, Y.; Ying, J. Y. J. Am. Chem. Soc. 2009, 131, 888-889. (37) Akbari-Birgani, S.; Hosseinkhani, S.; Mollamohamadi, S.; Baharvand, H. J. Biolog. Chem. 2014, 289, 16905-16913. (38) Pu, K.-Y.; Luo, Z.; Li, K.; Xie, J.; Liu, B. J. Phys. Chem. C 2011, 115, 13069-13075. (39) Samari, F.; Hemmateenejad, B.; Rezaei, Z.; Shamsipur, M. Anal. Methods 2012, 4, 4155-4160. (40) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer, Amsterdam, 2006 (41) Li, P.-H.; Lin, J.-Y.; Chen, C.-T.; Ciou, W.-R.; Chan, P.-H.; Luo, L.; Hsu, H.-Y.; Diau, E. W.-G.; Chen, Y.-C. Anal. Chem. 2012, 84, 5484-5488. (42) Huang, Z.; Pu, F.; Lin, Y.; Ren, J.; Qu, X. Chem. Commun. 2011, 47, 3487-3489. (43) Ma, Y.; Bai, H.; Yang, C; Yang, X. Analyst 2005, 130, 283–285. 16 ACS Paragon Plus Environment

Page 17 of 29

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(44) Wang, G.; Zhu, Y.; Chen, L.; Zhang, X. Biosens. Bioelectron. 2015, 63, 552-557. (45) Zhang, L.; Zhu, J.; Guo, S.; Li, T.; Li, J.; Wang, E. J. Am. Chem. Soc. 2013, 135, 24032406. (46) Sharon, E.; Freeman, R.; Willner, I. Anal. Chem. 2010, 82, 7073-7077. (47) Freeman, R.; Girsh, J.; Fang-ju Jou, A.; Ho, J.-a. A.; Hug, T.; Dernedde, J.; Willner, I. Anal. Chem. 2012, 84, 6192-6198. (48) Hami, Z.; Amini, M.; Ghazi-Khansari, M.; Rezayat, S. M.; Gilani, K. DARU J. Pharmaceut. Sci. 2014, 22, 30. (49) Zhang, W.; He, X.-W.; Chen, Y.; Li, W.-Y.; Zhang, Y.-K. Biosens. Bioelectron. 2011, 26, 2553-2558. (50) Ocaña, C.; Arcay, E.; del Valle, M. Sens. Actuators B 2014, 191, 860-865. (51) Ashe, D.; Alleyne, T.; Iwuoha, E. Biotechnol. Appli. Biochem. 2007, 46, 185-189.



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Figure Legends

Figure 1. (A) UV-vis spectrum of Cyt c (a) and fluorescence emission spectrum of Hb/AuNCs in 10 mM PBS of pH 7.4 upon excitation at 360 nm (b). (B) Schematic presentation of FRET from Hb/AuNCs to Cyt c. Figure 2. (A) Selectivity of the Hb/AuNCs toward different proteins, and hemin, ATP and GSH at 440 nm (λex= 360 nm); the concentration of each protein and hemin, ATP and GSH is 5 µM. (B) Time-dependent fluorescence intensity changes of the of the Hb/AuNCs for 10 µM of Cyt c. (C) Fluorescence emission spectra upon addition of different concentrations of Cyt c (0, 0.16, 0.32, 0.48, 0.70, 1.0, 1.5, 2.0, 3.0, 5.0, 7.0, 9.0, and 10 µM). The spectra were recorded at time intervals of 5 min. (D) Stern-Volmer plot of fluorescence quenching of the Hb/AuNCs by Cyt c. All experiments were carried out in 10 mM PBS of pH 7.4 with Hb/AuNCs concentration of 2.2 µM. Error bars represented as ±3σ. Figure 3. (A) (a) Excitation (λex = 470 nm) and (b) emission (λem = 530 nm) spectra of Cyt c1/AgNCs. (B) (a) Excitation (λex = 560 nm) and (b) emission (λem = 640 nm) spectra of Cyt c-2 /AgNCs. (C) Relative fluorescence intensity of oligonucleotide probes against different Cyt c concentrations. Figure 4. Schematics of PET analysis of Cyt c by DNA/AgNCs. Figure 5. (A) Time-dependent fluorescence intensity changes of the Cyt c-2/AgNCs probe in 1 µM of Cyt c. (B) Fluorescence response of Cyt c-2/AgNCs probe to 1 µM of Cyt c and other proteins Hb, BSA, HSA and Lys, Insulin and IgG as well as Hemin, ATP, GSH at 610 nm (λex= 560 nm). (C) Fluorescence emission spectra of Cyt c-2/AgNCs probe upon addition of different concentrations of Cyt c (0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.50, 0.55, 0.60, 0.65, 0.70, 0.80, 0.90, 1.0, 1.1, 1.3, 1.5, 1.75, 2.0, 2.5, 3.0, 4.0, and 5.0 µM). All spectra were recorded at time intervals of 20 min. (D) Stern-Volmer plot of fluorescence quenching of the Cyt c2/AgNCs probe by Cyt c. Inset shows the linear range from 0 to 1μM with LOD = 15.0 nM. The total experiments were carried out in PBS buffer (10 mM, pH =7.4) and Cyt c-2/AgNCs concentration of 25 µM. Error bars represented as ±3σ.


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Figure 6. (A) Fluorescence emission spectra of the Cyt c-2/AgNCs in the presence of variable concentrations of cancer cell extracts treated with (a) hypotonic buffer and (b) with CCLR buffer (both in the presence of 0, 3.0, 5.0, 7.0, 9.0, 11.0, 13.0, 15.0, 17.0, and 20 µL aliquots taken from initial 10000 cells mL-1 solution). The spectra were recorded at time intervals of 20 min. (B) Relationship between the fluorescence intensity and the cell extract (cells μL-1). Inset shows the linear range from 1 to 20 μL with LOD of 0.35 μL. (C) Time-dependent fluorescence intensity changes of the Cyt c-2/AgNCs probe in the presence of cell extract treated with hypotonic buffer (a) and with CCLR buffer (b) at constant concentration of 100 cells μL-1, by using 5 µL of cell extract. All experiments were carried out in 10 mM PBS of pH 7.4 and Cyt c-2 concentration of 25 µM. Error bars from three replicate experiments represented as ±3σ.


0.032 -2.4 µM

0.03-10 µM

0.32-9.6 nM

0.4-56.4 nM

1-10 µM

0.1-20 nM

Glutathione-capped CdTe quantum dots

PEGylated graphene oxide (GO) nanosheets/ fluorophore-tagged DNA aptamer Spirocyclic rhodamine B hydrazide/sodium dodecylbenzene sulfonate surfactant micelles Luminal/hydrogen peroxide Absorbance measurement of the Soret (Q) peak at 414 nm Aptamer-modified gold nanoparticles (apt-AuNPs)/ surface activated magnetic microparticles (MMPs)

Turn-Off ﬂuorescence

Turn-On fluorescence imaging

Turn-On ﬂuorescence

Chemiluminescence

Spectrophotometry

mass spectrometry (ICP-MS)

20

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5-600 µM

0-4.0 µM

0.4-1200 nM

0-0.5 mM

cytochrome c oxidase–DDAB (didodecyl-dimethylammonium) liquid-crystal film Hydroxylamine/Fe-Protoporphyrin IX/ Hg electrode Absorbance measurement in 436 nm Hb/AuNCs DNA/AgNCs

Square wave voltammetry

Single-sweep polarography

Immunohistochemistry


15.7

14.3

-

0.24

200

RSD: 2.1%, pH: 7.4, BE: Aptamer, Selectivity: Good, Time: 5, 20 min, RS: Apoptotic and nonapoptotic human embryonic kidney HEK293T cells, Recovery: 92.0-102.4%, Nanoplatform construction: One step for < 6 h (DNA/AgNCs) and 24 h (Hb/AuNCs), Label free, Applicable in drug screening.

Present Work

9

16

RSD: 1.65%, pH: 9.6, Selectivity: Good (except Hb, Mb, And HRP), Time: a few minutes, One step, RS: NR, Label based, Problems with mercury electrode is mercury toxic and slightly volatile. RSD: NR, pH: 7.4, BE: Antibody, Selectivity: NR, Time: NR, RS: Cardiomyocytes, Recovery: NR

51

1

15

50

RSD: NR, pH: 7.4, BE: Enzyme, Selectivity: NR, Time: NR, RS: Human serum, Recovery: NR, Nanoplatform construction: One step for > 24 h, Label based

RSD: 1.8%, pH: 7.4, BE: Antibody, Selectivity: Good (Hb and IgG), Time: 1h, RS: Apoptotic Hela cells, Recovery: NR, Nanoplatform construction: Multi step for > 4 h, Label free

RSD: 2.8%, pH: 7.0, BE: Enzyme, Selectivity: Fair ( Ascorbic acid (AA) and uric acid (UA), dialysis is needed in order to remove the interference due to AA), Time: NR, RS: Apoptotic human lung carcinoma A549 cells, Recovery: NR, Nanoplatform construction: Multi step for > 36 h, Label based , Applicable in drug screening.

RSD: 6.8%, pH: 7, BE: Aptamer, Selectivity: Rather good (Albomin, Fbr, IgG), Time:15 min, RS: NR, Nanoplatform construction: Multi step for >3 days, Label free

19

14

RSD: 2-3%, pH: 6.0, Selectivity: Good, Time: 20 min, RS: Rat liver mitochondria, Recovery: NR, high standard deviation is obtained with decreasing the sample size (16% for 1.27 pmol).

RSD: NR, pH: 7.4, BE: antibody & aptamer, Selectivity: Good, Time: 3 h, RS: apoptotic, HepG2 & R-HepG2 cells, Nanoplatform construction: Multi step for > 20 h, Applicable in drug screening

13

11

RSD: 2.26%, pH: 2.05, Selectivity: Good, Time: 12 min, RS: Cytosol fractions and mitochondrial extracts from control and digitalis-intoxicated guinea pig hearts, Recovery: 98.4-110.2%

RSD: 6.6%, pH: 7.4, BE: Aptamer, Selectivity: Good, Time: ~ 8h, RS: Human serum, Recovery: 87-97%, Nanoplatform construction: Multi step for ~ 3-4 days, this method not be able to detect Cyt c (10 pM) in serum sample.

10

12

RSD: < 3%, pH: NR, Selectivity: Good, Time: 30 s, RS: Human serum and pharmaceutical injections, Recovery: 98-108.8%, ,Label free RSD: NR, pH: 7.1, Selectivity: NR, Time: 1 or 2 h, RS: Isolated mitochondria from white Wistar rat hearts (Cardiomyocytes), Recovery: NR

8

17

RSD: NR, pH: 7.4, BE: Aptamer, Selectivity: Good, Time: > 30 min, RS: Cytoplasmic protein extract from nonapoptotic and apoptotic HeLa cells, Recovery: NR, Nanoplatform construction: Multi step for > 8 days, Label based, Cyt c assay was carried out by using Western blotting; Applicable in quantitative imaging.

RSD: 2.1%, pH: 8.0, Selectivity: Good (except proteins), Time:10 min, RS: NR, Label free

7

49

Ref.

RSD: 0.6%, pH: 9.0, Selectivity: Good (except Hb), Time: 10 min, RS: Synthetic & injection samples, Recovery: 95-106%, Nanoplatform construction: One step for >11 h, Label free

RSD: 2.2%, pH: 7.0, Selectivity: Good (BSA, Lys), Time: NR, RS: NR, Nanoplatform construction: Multi step for >12 h, Label based, this approach is still in its infancy, and has not been widely employed.

DLR=Dynamic linear range; DL= detection Limit; RS=Real Sample; BE= Biorecognition element; NR=Not reported; HQ: Hydroquinone; CcR: Cytochromecreductase; CNT: Carbon nanotubes; PPy: Polypyrrole ; GNP: Gold nanoparticle.

0-1.0 µM

0-10 µM

0.1-100 µM

Gold nanoparticle polydop-amine (AuNP/PDA) composites

Electrochemical impedance spectroscopy 30

500

1-1000 µM

CcR-CNT-PPy-Pt electrode CcR-GNP-PPy-Pt electrode

Electrochemical cyclic voltammetry

0.06

0.05-50 nM

Epoxy-graphite composite

0.8

Electrochemical impedance spectroscopy

0.8-80.6 nM

0.1 pmol

Antibody /micro-magnetic bead/ antigen/aptamer

0-40 pmol

0.06×10-3

0.03

NR

0.16

0.07

10

3.0

410

DL (nM)

Aptamer-based biobarcode (ABC)

HPLC

0.2-600 pM

0.97-24 µM

Molecularly imprinted polymer coated CdTe quantum dots


Capillary zone electrophoresis

DLR

Detection System

Method

Comment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1. Comparison of different Methods for the Detection of Cyt c

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Table 2. Determination of Cyt c in HEK293T Cells and Spiked Samples (n=3) treated with (A) CCLR Buffer and (B) Doxorubicin, using the DNA/AgNCs probe Treatment

Spiked amount (µM)

Found amount (µM)

A: CCLR

0 0.075 0.125 0.175 0.225

0.020 ± 0.003 0.089 ± 0.005 0.139 ± 0.003 0.199 ± 0.006 0.253 ± 0.002

0 0.5 1.5 2.5 3.5

0.29 ± 0.01 0.83 ± 0.01 1.71 ± 0.26 2.60 ± 0.23 3.61 ± 0.16

B: DOX

Recovery (n = 3) (%) _ 92.0 ± 3.7 95.2 ± 2.6 102.4 ± 3.4 103.4 ± 5.4 _ 108.4 ± 0.8 94.8 ± 8.3 92.4 ± 5.0 94.8 ± 4.1



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