Silicon Quantum Dot-Based Fluorescent Probe: Synthesis Characterization and Recognition of Thiocyanate in Human Blood.

Allylamine-functionalized silicon quantum dots (ASQDs) of high photostability are synthesized by a robust inverse micelle method to use the material as a fluorescent probe for selective recognition of thiocyanate (a biomarker of a smoker and a nonsmoker). The synthesized ASQDs were characterized by absorption, emission, and Fourier transform infrared spectroscopy. Surface morphology is studied by transmission electron microscopy and dynamic light scattering. The synthesized material exhibits desirable fluorescence behavior with a high quantum yield. A selective and accurate (up to 10-10 M) method of sensing of thiocyanate anion is developed based on fluorescence amplification and quenching of ASQDs. The sensing mechanism is investigated and interpreted with a crystal clear mechanistic approach through the modified Stern-Volmer plot. The developed material and the method is applied to recognize the anion in the human blood sample for identification of the degree of smoking. The material deserves high potentiality in the field of bio-medical science.

Introduction

Quantum dots (QDs) are highly photostable nanosized (2–10 nm)[1] semiconductor having unique fluorescent property, which is due to the quantum confinement effect.[2,3] It has a broad range of application in the field of bioimaging,[4] optoelectronics,[5] photonics,[6] biosensing,[7] and chemical sensing.[8,9] Among the QDs searched so far (CdSe, CdTe, CdS, ZnS, ZnSe, PbS, PbSe, GaAs, GaN, InP, and InAs), silicon QDs (SQDs) are one of the best candidates for fluorescent probe because of low toxicity as well as high photostability.[3,10] Apart from noncytotoxicity, SQDs also has tunable fluorescence property, high luminosity,[11] and less photobleaching effect[12] in comparison with other organic dyes. Moreover, silicon is more abundant compared to others. Therefore, SQDs are a very interesting moiety for research.

Various physical and chemical processes such as electrochemical etching,[13,14] sol–gel method,[15,16] laser ablation,[17,18] thermal annealing, thermal vaporization,[19] and inverse micelle[20−22] are available for the synthesis of the SQDs. Among these, though the inverse micelle is robust one, it has two major drawbacks: (i) easy aerial oxidation at room temperature and (ii) water insolubility. Water dispersibility is one of the most important and essential features required for its analytical and biological research.[23] Therefore, surface modification is strictly recommended for prevention of areal oxidation as well as the incorporation of water solubility. Surface modification is done by various materials such as acrylic acid,[24] propionic acid,[25] vinyl pyridine,[26] and allylamine.[22] Recently, the last one is getting more importance[22,23,27] because of its structural features. Allylamine moiety possesses the carbon–carbon double bond and the amine group in terminal positions, which are suitable for hydrosilylation[28] in the presence of a platinum catalyst at the room temperature. The surface modifier has some effect on the fluorescence property.[23] It is found that amine-terminated SQDs are more effective in exhibiting intense fluorescence in comparison to alkyl groups.[23,27] Thus, allylamine emerges as the most important surface modifier.

Low-level thiocyanate is an important constituent in the human body; but its high concentration exhibits an evil effect. Clinical studies show that the concentration of it in the blood serum becomes high for the smoker in comparison to the nonsmoker.[29,30] The concentration of the anion present in the human plasma level found to be in the ranges 9–12 and 2–3 mg/L for the smoker and the nonsmoker,[29,30] respectively. The half-life period of thiocyanate in the human blood is 1–2 weeks.[31] Therefore, the thiocyanate ion may be used as a biomarker for smokers and nonsmokers.[32]

During smoking, hydrogen cyanide is formed because of pyrolysis or combustion of nicotine present in tobacco. The inhaled cyanide is detoxified into the thiocyanate through incorporation of mitochondrial rhodanese and β-mercaptopyruvate (shown in Scheme ).[33] The high concentration of the thiocyanate ion is not only the strongest atherogenic factor but also liable for the formation of atherosclerotic plaques within coronary arteries,[34] which increases the risk of cerebral infarction stroke.[35,36] It is also responsible for increasing the plasma myeloperoxidase level for which protein dialysis may be hampered along with uraemic complication.[36] Again, the anion is remarkably responsible for blocking of the iodide uptake into the thyroid gland. This is highly dangerous for children and pregnant women because of the decrease in thyroxin formation and iodide deficiency.[37]

Scheme 1

Detoxification of CN– in the Human Body

Among the available methods till date for the thiocyanate sensing, flurometric is the most scientifically convincing, easy, and less time-consuming process. Toida et al.[38] reported fluorometeric sensing of the thiocyanate ion using Konig reaction, but a major bottle neck of the method is that there is no concentration-dependent study. Sarkar and his coworker[39] studied the same anion sensing using the Cd(II) complex. However, the method is not a robust one. Zhang and his group[40] synthesized gold nanoparticles and carbon dot-based probes for thiocyanate ions. As per our best knowledge and literature survey, till date, no report is found for allylamine-functionalized silicon QD (ASQD)-based thiocyanate sensing.

Here, we reported for the first time robust, quantitative, and selective flurometric sensing of thiocyanate ions (a biomarker of smokers and nonsmokers) with the designed and developed ASQD. The indigenous material and method has been tested in real sample analysis.

Result and Discussion

Characterization of the ASQDs

UV–Vis Absorption Spectral Study

A steady-state UV–vis spectrum was recorded to study the optical properties of ASQDs (Figure ). It showed a continuous absorption pattern from 500 to 200 nm with a prominent shoulder around 327 nm. The appearance of the shoulder is a distinctive nature of SQD because it is the corollary of well-studied Γ to Γ direct band gap transition.[41] Therefore, the nature and position of the shoulder in the absorption spectra confirmed that ASQDs were synthesized successfully.

Figure 1

Steady-state normalized absorption spectrum of ASQD, H-Terminated Silicon QD (HSQD), and allylamine.

Fluorescence Study

The steady-state fluorescence spectrum of aqueous ASQDs recorded in ambient temperature is shown in Figure . It was found that the material exhibited emission maximum (λmax) at 439 nm on excitation at 327 nm. The emission of the QDs occurred probably because of the jump of the promoted conduction band electron to the valence band.[42] Thus, the fluorescent spectra revealed the successful formation of ASQDs.

Figure 2

Steady-state normalized emission spectrum of ASQDs and HSQDs.

Fourier Transform Infrared (FTIR) Study

The FTIR spectrum of ASQDs is displayed in Figure . The appearance of a band along with an overtone at 3124 cm–1 (νN–H stretch) and a sharp peak at 1569 cm–1 (νN–H bending) indicated the presence of a primary amine group in ASQD.[43] The spectrum also exhibited a band at 1185 cm–1 because of the C–N stretching and another two strong sharp peaks at 1500 and 1396 cm–1 for the vibrational scissoring and symmetric bending of Si–CH2[44] moieties, respectively. Furthermore, the appearance of a small peak at 1662 cm–1 indicated successful attachment of allylamine in the silicon surface.[20,44] Hence, FTIR spectroscopy revealed that ASQDs were synthesized successfully.

Figure 3

FTIR spectrum of the ASQDs.

Morphological Analysis

The morphological analysis of the synthesized ASQDs was carried out by the assistance of transmission electron microscopy (TEM) (A, B), selected area electron diffraction (SAED) (C) and dynamic light scattering (DLS) (D) study. The TEM image of ASQDs (Figure A) reveals that the synthesized QDs were homogeneously dispersed throughout the matrix with almost a spherical shape. It is also evident from the image (Figure A,B) that the size of the QDs was uniformly distributed around the 1–2 nm range. The presence of prominent lattice fringe and a circular ring pattern in the TEM and SAED images (Figure C), respectively, is a sign of the crystalline nature of QDs.

Figure 4

TEM images (A,B), SAED pattern (C), and DLS data (D) of ASQDs.

The DLS study (Figure D) shows that the average size of the QDs was around the 5 nm range. However, the result obtained from the DLS study does not resemble with TEM data (1–2 nm range). This is due to the fact that DLS considers the hydrodynamic size distribution, which is larger than the consideration by TEM. Because the ASQDs were terminated with a hydrophilic group (allylamine), the results obtained by DLS are quite reasonable.

Therefore, homogeneous dispersion and uniform size distribution from morphological studies indicate that the synthesized ASQDs are of high quality.

Quantum Yield

The fluorescence quantum yields (φf) of ASQDs in water were calculated with respect to 1-methylindole (in aqueous medium = 0.33 and fluorescence excitation wavelength, λ = 280 nm) at 296 K[45] by the following equation.[46]where, φf, If, A, and n represent the fluorescence quantum yield, intensity, absorbance, and refractive index of the solvent, respectively, for the sample under investigation, while φfR, IfR, AR, and nR designate the fluorescence quantum yield, intensity, absorbance, and refractive index of the solvent for reference material, respectively. Both the sample and the reference are excited at the same wavelength and the A, AR values are kept very low (<0.05) to measure If and IfR, respectively. The obtained value of φf for ASQDs in water was found to be 0.37.

Sensing of Thiocyanate Ions

Photoluminescence (PL) Titration of ASQDs at Various Concentrations of SCN– Ion

The PL spectra of ASQDs were carried out in the absence and presence of ammonium thiocyanate (NH4SCN). The concentration of thiocyanate was varied from 1 × 10–10 to 15 × 10–1 M. The reason for taking the lower concentration of 1 × 10–10 M was that the system could not respond behind it; thus, this level of concentration is termed as the limit of detection (LOD). The emission spectrum of pure ASQD consists of a sharp peak at 439 nm and a weak band in the region 750–900 nm. The PL intensity at 439 nm was amplified slightly with the gradual addition of the thiocyanate anion up to 1 × 10–6 M (critical point) and then quenched steeply with further increase of the anion (Figure b). A similar trend was observed for the weak band in the longer wavelength region (750–900 nm) (Figure a inset). The origin of the PL emission of ASQDs is a complicated combination of direct (quantum confinement effect) and indirect band gap transition.[20] Zhou et al.[47] suggested that 1–2 nm SQDs (silicon QD) with hydrogen or carbon surface termination have direct band gap optical transitions, which is attributed to PL at the UV–visible region in the electronic spectra. However, Wang et al.[48] suggested that in ASQDs, there should be a photoinduced charge transfer (CT) between Si- and N-terminated allylamine groups because of electronegativity difference. The resulted excitonic state of ASQDs due to CT was responsible for its PL emission. The initial minor amplification of PL intensity may be attributed to the stabilization of the excitonic state of ASQDs in the presence of the low level of the thiocyanate ion (through hydrogen bonded interactions). However, the steep decrease of PL intensity beyond the critical point may be explained in terms of photoinduced electron transfer (PET) from the thiocyanate ion to the excitonic state of ASQDs. This interaction seemingly affects the exciton dipole moment of ASQDs, so as to reduce its radiative transition probability, thereby reducing the emission intensity of the dots.

Figure 5

(a) PL titration of thiocyanate ions with ASQDs (inset: the expansion of the near-IR region); (b) PL intensity at 439 nm at various concentrations of the thiocyanate ion.

The observed dynamic fluorescence quenching in the present work may be due to several processes such as PET, photosensitized excitation energy transfer (PEET), and so forth. However, thiocyanate ions showed very weak radiative transition (absorption) from the ground state (S0) to the first excited singlet state (S1) and no radiative transition (fluorescence emission) from S1 to S0. The spectral overlap between the absorption spectra of thiocyanate ions and the fluorescence emission spectra of ASQD is negligible. Therefore, we can rule out the possibility of PEET from the singlet-excited ASQD to the ground-state thiocyanate ions. This clearly indicated the presence of the other dynamic quenching process, that is, PET from the ground-state thiocyanate ions (electron donor, D) to the singlet-excited ASQD (electron acceptor, A), being responsible for the observed fluorescence quenching of the singlet-excited ASQD in the presence of ground-state anions. It may be noted that PET between the D–A system may occur either by exciting the donor (in the presence of the ground-state acceptor) or by exciting the acceptor (in the presence of the ground-state donor).[46]

Modified Stern–Volmer (SV) Plot

An attempt has been taken to explain the quenching phenomena by the conventional Stern–Volmer (SV) plot using eq .where F0 and F represent the fluorescence intensity of the fluorophore ASQD in the absence and presence of the quencher thiocyanate ions, respectively, and KSV is the SV constant (or dynamic quenching constant) and [Q] is the quencher concentration. The SV plot (Figure ) showed a downward curvature toward the X-axis. Thus, it is clear from the nonlinearity of the plot that quenching data did not follow the usual SV equation (eq.). Therefore, another attempt was designed to explain the quenching through the modified SV plot (Figure ).

Figure 6

SV plot of the ASQD in the presence of different thiocyanate ion concentrations.

Figure 7

Modified SV plot of ASQD.

Midoux et al. found a complex SV plot for the fluorescence quenching of tryptophan by trifluoroacetamide (TFA) and proposed that the complex SV plot may be attributed to the presence of two fluorophore populations of tryptophan, one of which is not accessible to the quencher.[49] This kind of a different accessibility of the fluorophore for fluorescence quenching has also been found for the quenching of lysozyme by iodine.[50] A modified SV equation has been developed to explain this particular phenomenon:[46]Here, the subscript “0” refers to the fluorescence in the absence of the quencher; the subscripts “a” and “b” stand for two populations of fluorophores, that is, accessible and inaccessible or buried, respectively. In the presence of the quencher, the fluorescence intensity of the accessible fraction decreases according to the normal SV equation (eq ), while the inaccessible fraction is not quenched. As a consequence, the observed fluorescence intensity may be written as follows.Here, Ka is the SV quenching constant of the accessible fraction. From eq and 3, we get the following relation.

Inversion of eq followed by the division into eq gives the following relation.Here

Applying this modification eq , a plot of F0/ΔF versus 1/[Q] for the present fluorophore-quencher system yielded a straight line (Figure ), which gives rise to the quenching constant (Ka). The value of Ka was found to be 19.79 L mol–1, which indicated appreciable binding between the fluorophore-quencher.

Selectivity Study

The selectivity study of the ASQDs (Figure A) toward the thiocyanate ion was done by taking 0.01 (M) concentration of different anions such as SO4–2, SO3–2, NCO–, and NO3–. The different anions were mixed with a fixed amount of (0.2 mL) ASQDs, and the mixture was subjected to a PL spectrometer. The spectra of different anions were compared to the spectra of the pure ASQDs. It was observed that there was no significant change in spectra (i.e., almost unaffected) with respect to the pure ASQDs spectrum for all the anions studied except the thiocyanate ion. The spectrum of the thiocyanate ion was quenched significantly. Moreover, to check whether there are any effects of the cationic part of the thiocyanate, potassium thiocyanate was taken with four different concentrations. Interestingly, the spectral behavior (Figure B) matched with the spectra of ammonium thiocyanate (Figure a), which indicated no role of the cationic counterpart in the thiocyanate sensing process. Therefore, it may be concluded that ASQDs can sense thiocyanate ions selectively.

Figure 8

(A) Emission intensity of ASQDs toward various anions, (B) PL spectra of ASQDs at different KSCN concentrations (where A1, A2, A3, A4, and A5 are 10–1, 10–3, 10–5, 10–7, and 10–9 (M) KSCN concentrations, respectively).

Thiocyanate Recognition in the Blood Sample

The thiocyanate sensing ability of ASQDs was not limited in synthetic solutions, but it was also carried out in real biological samples (Figure ). The results showed that the photoinduced quenching effect was more significant for the smoker blood samples (S1, S2, and S3) compared to the nonsmokers (NS1, NS2, and NS3). Thus, the synthesized material ASQD can acts as a biomarker toward the blood of smokers and nonsmokers by differential quenching of fluorescence intensity. The developed quenching method also revealed that the smoker blood contains more thiocyanate than its nonsmoker counterpart.

Figure 9

PL spectra of ASQD in the smoker and the nonsmoker blood sample with an error bar (error is almost equal to 5%) (where NS and S stand for nonsmoker and smoker, respectively).

Now, for searching the quantitative degree of smoking, a standard curve (PI vs various synthetic solutions containing blood serum and a known amount thiocyanate within 2–12 mg/L) has been drawn. Comparing the results of real samples with the standard curve, it may be concluded that the degree of smoking was the highest for sample S3 (∼11 mg/L) and the lowest for S1 (∼9 mg/L). On the other hand, the blood serum of nonsmokers contained thiocyanate with the range 2–3 mg/L.

Conclusions

Highly photostable ASQDs of desired size (1–2 nm) and distribution (around 5 nm range) is synthesized by a robust inverse micelle method.

(ii)The synthesized material shows characteristic absorbance at 327 nm and exhibits unique fluorescence of a sharp peak at 439 nm and a weak band in the region 750–900 nm with high quantum yield (0.37).

The quenching phenomena of ASQDs with thiocyanate ions are well explained through the modified SV plot. The value of the quenching constant (Ka) was found to be 19.79 L mol–1, which indicates appreciable binding between the fluorophore and the quencher.

The developed ASQDs recognize thiocyanate ions selectively and accurately with the LOD value of 1 × 10–10 M.

The explored recognition method could estimate thiocyanate ions in the human blood sample to identify the degree of smoking. Blood of the smoker contains a high amount of thiocyanate ions (9–12 mg/L) compared to that of the nonsmoker (2–3 mg/L). Thus, ASQDs may be useful as a biomarker for the smoker and the nonsmoker.

The material and the method deserve high potentiality to be used in the field of biomedical science.

Materials, Instruments, and Method

Materials

Tetraoctyl ammonium bromide (TOAB), silicon tetrachloride (99%), lithium aluminum hydride in tetrahydrofuran (LAH in THF), and chloroplatonic acid hexa-hydrated in Isopropanol were purchased from Sigma-Aldrich. Toluene (99%), methanol, allylamine, ammonium thiocyanate, potassium thiocyanate, phosphate-buffered saline (PBS) buffer, and ammonium chloride were purchased from the Marck, Mumbai, India. Double-distilled water obtained from the water double distillation set. After distillation, toluene and methanol were used after distillation.

Instrumentation

A sonicator of 40 kHz (Branson-1510) was used for sonication. All the electronic absorption spectra were measured in the steady state of the synthesized ASQDs by using a JASCO V-650 absorption spectrophotometer with a 1 cm path length rectangular quartz cuvette at ambient temperature (300 K). All the PL spectral measurements were done in the steady state by using a JASCO FP-6500 fluorescence spectrometer, and the sample was taken in a rectangular quartz cuvette (1 cm path length) at room temperature (300 K). The solvent was evaporated by using a Superfit, Rotavap rotary evaporator. Emission was recorded at right angles of the excitation light direction in to avoid stray light. A Shimadzu-8400S spectrometer was used for FTIR spectra. The sample for the FTIR spectra was prepared in KBr pellets. TEM and SAED studies were carried out by using a JSM-100CX, Jeol TEM microscope. The sample for TEM analysis was prepared by putting of one drop sample in a standard carbon-coated copper grid and dried overnight in a vacuum oven. The DLS study of the ADSQ was done for size distribution analysis by using a Malvern Zetasizer Ver.6.34 instrument. Ultra centrifuge was used for the collection of the blood serum from blood samples.

Synthesis

Synthesis of HSQDs

The methodology follows the standard synthetic procedure of silicon QDs by inverse micelle[20,22] with minor modification (Scheme ). In brief, TOAB (0.003 mol) was dissolved in dry toluene (100 mL) with the help of a magnetic stirrer. Then, 92 μL of SiCl4 was added thorough a gas-tight syringe, and the reaction mixture was allowed to mix well for 1 h in inert atmosphere. A clear and transparent solution was obtained. A calculated amount of LAH was mixed with that transparent solution to obtain the desired mole ratio of SiCl4/LAH (1:21.7). The reaction mixture became opaque at once, probably because of the micelle formation. Now, the reduction of SiCl4 was allowed to continue within the micelle for 3 h. After that, a stoichiometric amount of anhydrous methanol was added to change the polarity of the medium and to remove the unreacted LAH. The change in polarity probably destroys the micelle as evident from the appearance of clear solution. The clear solution mainly contained HSQDs.

Scheme 2

Synthesis of HSQDs and ASQDs

Synthesis of ASQDs

The surface functionalization of HSQDs was carried out to produce ASQDs through Chalk–Harrod hydrosilylation reaction with some modification.[28] A calculated amount of the platinum catalyst and allylamine reagent was added to maintain the designed stoichiometric volume ratio of catalyst/allylamine (1:20). The solution was then allowed to react for more than 3 h in ambient temperature. Then, all the solvents were removed by rotary evaporation, which leads to the formation of white dry powder. The dry powder mainly consisted of ASQDs and the unreacted surfactant. To remove the surfactant, it was dispersed into 20 mL of distilled water followed by sonication at 40 kHz for 30 min. Then unreacted TOAB was removed by successive filtration through a 0.22 mm membrane. The filtrate contained mainly aqueous solution of ASQDs.

Sample Preparation

Recognition of Thiocyanate in Synthetic Solutions

The recipe for the preparation of synthetic solutions is depicted in Table . The thiocyanate solution in PBS buffer (2.3 mL) was placed in series of sample vials by varying its concentration from the molar to decinanomolar range at a fixed amount of ASQDs (0.2 mL of 8.05 × 10–6 M). Each synthetic solution was subjected to fluorometric analysis. The selectivity of ASQDs toward thiocyanate was checked with different synthetic solutions such as sodium sulfite, sodium isocyanate, sodium nitrate, ammonium chloride, and magnesium sulfate. The synthetic solutions in PBS were prepared following the same procedure as thiocyanate.

Table 1

Recipe for the Preparation of Different Thiocyanate Solutions

no. of synthetic solutions	concentration of thiocyanate in PBS buffer (M)	volume of thiocyanate (mL)	volume of 8.05 × 10–6 (M) ASQDs (mL)
Apure	nil	nil (only 2.3 mL PBS buffer)	0.2
A1	15 × 10–1	2.3	0.2
A2	5 × 10–1	2.3	0.2
A3	1 × 10–1	2.3	0.2
A4	1 × 10–2	2.3	0.2
A5	2 × 10–2	2.3	0.2
A6	3 × 10–2	2.3	0.2
A7	4 × 10–2	2.3	0.2
A8	1 × 10–3	2.3	0.2
A9	1 × 10–4	2.3	0.2
A10	1 × 10–5	2.3	0.2
A11	1 × 10–6	2.3	0.2
A12	1 × 10–7	2.3	0.2
A13	1 × 10–8	2.3	0.2
A14	1 × 10–10	2.3	0.2

Recognition of Thiocyanate in the Blood Serum

A series of freshly collected 5 mL human blood samples of both smokers and nonsmokers were taken from Pearson Memorial Hospital, Visva-Bharati, Santiniketan-731235, India, and placed in centrifuge tubes. After 30 min, the samples were subjected to centrifugation, and the blood serums were collected carefully. Then, blood serum (2.3 mL) and ASQDs (0.2 mL) were mixed thoroughly for the fluorescence study.