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Enhanced intrinsic fluorescence (~x103) from novel carboxidized nanosculptured thin films (CO-nSTFs) of silver is reported. The sources of intrinsic fluorescence, confirmed by X-ray photoelectron spectroscopy, are Ag2O grains and residual carbon formed on the outer layer of silver nSTFs when exposed to air, while the localized surface plasmons on silver nSTFs enhance this intrinsic fluorescence. The CO-nSTFs are optimized with respect to porosity for the maximum enhancement. A sensor developed by using the self-assembled monolayer technique on optimized CO-nSTF is used for the label free detection of glycated hemoglobin, performed by simultaneously using fluorescence imaging and spectroscopy. The specificity of the sensor is established from control experiments on hemoglobin. These novel nanorod like intrinsically fluorescent CO-nSTFs pose huge potential in label free biosensing, light sources, imaging and many more applications.
© 2017 Optical Society of America
1. Introduction
Fluorescence has fascinated researchers for more than 40 years as a powerful tool for studying various systems, especially in the field of life sciences, where it has been employed for studying protein structures, imaging, drug delivery, manipulation of sub-cellular structures, protein-ligand interactions, etc [1–5]. Apart from life sciences, fluorescence based techniques have been utilized in memory devices, light emitting diodes, lasers, molecular rulers, etc [6–9]. Wide range of applications of fluorescence have been reported in the fields of sensing, where detection of analytes of clinical importance, such as cancer biomarkers, etc. was reported [10–14]. When it comes to detecting fluorescence from single molecules, among the techniques employed, plasmon enhanced fluorescence has proven to be one of the best candidates [15,16]. When a fluorescent molecule is brought close to a plasmonic structure, its optical emission is found to enhance manifold [17,18]. The enhancement of the signal from molecules near a plasmonic structure is attributed to the highly amplified electromagnetic fields in the vicinity of the metallic nanostructures due to localized or propagating surface plasmons [19,20]. Such a scheme has mostly been utilized in surface enhanced spectroscopies such as surface enhanced Raman spectroscopy (SERS) and surface enhanced fluorescence (SEF) which were found to be quite useful for applications in various fields that depend on the enhancement of the emitted signals from the molecules present in the vicinity of nanostructured plasmonic surfaces [16,21–24]. Nano-sculptured thin films (nSTFs) of silver were found to enhance SERS and fluorescence signals from 4-Aminothiophenol (4-ATP) and Rhodamine123 molecules respectively [25]. The nSTFs are the porous films with quite fascinating optical properties and have been demonstrated for various applications [26]. For the specific detection of biomolecules of interest, biomolecular recognition layers of analyte specific ligands are, in general, immobilized on the surface of nSTFs [27]. In most of the cases, entities such as antibodies, enzymes or other bio-recognition elements (BREs) (e.g. bacteriophages) are cross-linked to the metal surface via a suitable cross-linker [28,29]. The capture of the specific analyte by the BREs results in a modulation of the SERS/SEF signal, which can quantitatively be translated into the concentration of the analyte in a given sample. Further, the specificity of such sensors is ensured by the BREs and anti-fouling agents which are immobilized on the sensor surface to avoid any non-specific binding over it. However, the electromagnetic fields being highly localized near the metallic nanostructures, the enhancement of the Raman/fluorescence signal is only possible when the molecule of interest is placed in the nanoscale vicinity of the metal surface. Therefore, SERS/SEF signals from an analyte molecule can specifically be acquired only if: (1) they are very small in size, (2) the cross-linker + BRE length is small. To say, the localized field of the plasmonic architecture must reach well within the analyte to enhance the Raman/fluorescence signal from it. In most of the sensors relying on the fluorescence technique, a fluorescent label must be tagged to the analyte molecule [23,30–33]. Since the enhancement is achieved in a small distance from the surface of the nanostructure, the fluorescence signals from labels cannot be amplified strongly, as in most of the fluorescence based assays, the labels are tagged in the end and are generally far away from the surface.
Fluorescence signals from nanodots of silver with sizes smaller than 20 nm were reported and used for memory applications [34–36]. However, the fluorescence in bigger Ag crystals was not observed [37]. The fluorescence in small nanodots was attributed to the formation of silver oxide Ag2O, while the bigger nanodots did not show fluorescence because they were not fully oxidized. It was further observed that carbon nanodots exhibit enhanced fluorescence when brought in contact with plasmonic structures [38,39]. Such schemes can be combined to produce chips with enhanced intrinsic fluorescence, as carbon nanodots as well as Ag2O both possess small fluorescence. If Ag2O and carbon nanodots can somehow be attached to plasmonic structures, they might be useful sources of enhanced fluorescence, because of electromagnetic enhancement. Such a structure with enhanced intrinsic fluorescence can be utilized in various potential applications such as optical memory, imaging, label free biosensors, etc. In the present study, we utilize the aforementioned idea to assess enhanced fluorescence signal. Gold (Au) is generally preferred over silver for biosensing applications because of its low reactivity and stability. However, certain applications prefer Ag over Au because of its high plasmonic quality factor [40]. In the present study, Ag was preferred over Au both because of its ability for the oxidation, which leads to intrinsic fluorescence and relatively higher quality factor of plasmon resonance, which leads to high plasmonic enhancement.
Diabetes is one of the major health concerns worldwide and is monitored majorly by measurement of immediate fasting glycemic index of glucose either in the saliva or blood plasma. However, sometimes the measurements might be deceiving, because they can fluctuate due to improper metabolism of blood sugar at different times of the day. Glycated hemoglobin (HbA1c) results from the glycosylation of hemoglobin (Hb) in the blood and for amounts of sugar larger than that of allowed physiological range in the blood, it has elevated levels, which again is a marker for elevated glucose levels [41]. HbA1c presents a more reliable measure for diabetes because of its constant levels of glycemic index over a period of 3 months and hence has become one of the important tests for confirmation and control of diabetes. The allowed levels of HbA1c in human blood generally range between 4 and 8% of total human Hb [42].
In the present study, the findings of novel CO-nSTFs (CO stands for Carbon-Oxygen or Carboxidized) of silver which possess enhanced intrinsic fluorescence and their use in label free biosensing have been reported. The reasons of intrinsic fluorescence and its enhancement were confirmed via X-ray photoelectron spectroscopy (XPS). The CO-nSTFs were optimized with respect to porosity and a sensor for HbA1c detection was developed on the optimum structure. The sensor was developed over 4-ATP layered CO-nSTF surface by immobilizing a self-assembled monolayer (SAM) of anti-HbA1c (anti-glycated hemoglobin) antibody over it. Further, the nonspecific binding sites were blocked by bovine serum albumin (BSA) to prevent the occurrence of any false signals. Specific detection of HbA1c was demonstrated as compared to Hb. The beauty of this scheme is that this sensor relies on a kind of SEF platform and does not require any fluorescent tags.
2. Enhanced intrinsic fluorescence
A fluorescence microscope (Olympus, BX51) was integrated with a spectrometer to record the fluorescence images and spectra from a micro-spot of the same chip at the same time. A schematic of the experimental setup is shown in Fig. 1. Light from a mercury arc lamp was first passed through a diffuser and then collected by lenses, and then collimated and further passed through UV and neutral density filters when required. For the excitation of fluorescence using the mercury green light an excitation filter around 546 nm was employed. This was realized by using filter cubes, as shown in the Fig. 1. The filter cube comprises of a green excitation filter (546 nm Hg line), a dichroic splitter and an emission filter (590 nm) to block the parts of the excitation light, which might reflect back from the sample. The fluorescence light collected from a micro-spot of the sample was divided into two parts; one going to the camera for recording of the image, and the other through the eyepiece to a fiber optic spectrometer for recording the spectrum. The fluorescence image from a micro-spot of a CO-nSTF chip with 30% porosity is shown in Fig. 2(a). A very bright fluorescent image from the chip could be captured. This is the direct evidence of enhanced intrinsic fluorescence from the chip. Figure 2(b) shows the schematic of enhanced intrinsic fluorescence from a nanorod of the CO-nSTF chip. The origin of this fluorescence can be attributed to the formation of Ag2O and residual carbon on the outer layer of the Ag nSTF, when exposed to air. This fluorescence is further enhanced by the electromagnetic enhancement due to localized surface plasmons on the nanorods and the gap plasmons in the voids.
Fig. 2 (a) Intrinsic fluorescence image of CO-nSTF with 30% porosity (b) Schematic of enhanced fluorescence from one nanorod of CO-nSTF.
To prove our arguments on the source of the intrinsic fluorescence, we present ahead X-ray photoelectron spectroscopy (XPS) measurements of the composition of the few nanometers of the surface of the CO-nSTFs. Figure 3(a) shows the XPS survey spectrum of the CO-nSTF, which shows multiple peaks. These peaks correspond to various elements present in the CO-nSTF. To understand the contribution of the various constituents, highly resolved XPS spectra were recorded in the region of the observed peaks and matched with the reference database. Figures 3(b)-3(e) represent the XPS spectra recorded around the peaks observed in Fig. 3(a). It can be seen that, when matched with the reference database, the peaks observed in Figs. 3(b)-3(e) correspond to Si, C, Ag and O respectively. Because of the porosity of the film, we get some fraction of the Si originating from the substrate as well. The percentage composition of these constituents in the CO-nSTF film has been tabulated in Table 1. The observation of carbon and oxygen in the XPS measurements confirms the existence of Ag2O and residual carbon in the upper crust (or say carbo-oxidation) of the nSTFs. The low amount of Ag2O does not change the peak shape of the Ag 3d peak remarkably in comparison with pure Ag. But the low energy component of the O 1s signal at 530.94 eV is a clear indication for a Ag–O bonding. The occurrence of fluorescence from oxidized Ag nanoclusters and complexes of Ag nanoparticles with carbon quantum dots (Ag/CQDs) were reported earlier by a few groups as well, which further strengthens the concept of intrinsic fluorescence from CO-nSTFs [43]. Furthermore, the small peaks in the SEF spectra at around 640nm and 650 nm may be attributed to the two Raman peaks observed by Yang et.al. from Ag/CQDs at around 1360 cm−1 and 1540 cm−1 respectively known as the D and G bands of graphite. The D band is associated with the vibrations of C atoms having dangling bonds in the plane terminations of disordered graphite or glassy carbons while the G band corresponds to the E2g mode of graphite related to the vibration of sp2 - bonded C atoms in a two-dimensional hexagonal lattice. The intensity ratio between the two bands (ID/IG) is a measure of the disorder extent of carbon materials, as well as the ratio of sp3 /sp2 carbons. The CO-nSTFs were then etched about 10 nm in the XPS chamber using 1000 eV Ar ions and the XPS measurements were performed after etching. The XPS survey spectrum after etching is presented in Fig. 3(f), while the O1s scan in the inset. It can be observed that almost no peaks for O and C are observed after etching, as compared to that in Fig. 3(a). A zoomed in look on O1s scan, presented in the inset of Fig. 3(f) shows very nominal/ small fraction of O as compared to the intact CO-nSTFs as presented in the Fig. 3(e). This control experiment confirms the existence of oxygen and carbon only in first few nanometers of the upper layer of the CO-nSTF.
Fig. 3 XPS scans for (a) all the constituents –survey scan, (b) Si -, (c) C -, (d) Ag -, (e) O - in the CO-nSTF with 30% porosity, (f) CO-nSTF after etching about 10 nm of the film. Inset shows the O1s scan counts as compared to that in Fig. 3(e).
Table 1. Elemental ID and quantification from XPS measurements
To understand the intrinsic fluorescence more quantitatively and optimize the structure and its performance, the fluorescence spectra as well as the images from the CO-nSTFs of different porosities were thoroughly studied. We have plotted ahead the fluorescence spectra recorded from the CO-nSTFs of different porosities in Fig. 4(a). A sharp drop in the fluorescence power around 590 nm, while blocking the blue side of the spectrum is observed due to the band edge of the long pass emission filter. However, for the present study, the 590 nm band edge can be considered as the point of interest. It can also be observed that the CO-nSTFs with different porosities possess different intrinsic fluorescence intensities and the CO-nSTF with about 30% porosity has the maximum intrinsic fluorescence in our experimental window. A closer look on the Fig. 4(a) reveals that with an increase in the porosity from 0 to 60%, the intrinsic fluorescence first increases up to 30% porosity and then shows a decrease with further increase in porosity from 30 to 60%. Hence, ~30% porosity can be considered to be the optimum for the enhancement. The reason for 30% porosity to be the optimum can be given similar to what was explained elsewhere following our optimization of n-STFs for SERS [25]. Briefly, the enhancement due to the nSTFs comes from the enhanced plasmonic fields which arise due to localized surface plasmons on the nano-columns. When the porosity is small, the number of nanocrystals of Ag2O + residual C (let’s call them AgCO complexes) in the gap between the nano-columns is small. However, at the same time, the field in the gap region is relatively larger than that on the nano-columns. Hence, the overall enhancement is not the optimum. When the porosity in increased, the number of AgCO complexes in the gap increases, but the field decreases. Hence, there is a trade-off between the porosity and the enhancement. Around 30% porosity, the enhancement contributed by the AgCO complexes in the gap and that due to the nano-columnar region add to give the maximum enhancement.
Fig. 4 (a) Intrinsic fluorescence spectra for varying porosity of CO-nSTFs, (b) Surface enhanced fluorescence spectra from spin coated rhodamine123 over nSTFs of varying porosity.
Further increase in the porosity leads to de-coupling of the plasmonic field of the nano-columns in between the gaps. Hence, even though the number of AgCO complexes in the gaps increases with porosity, the overall enhancement does not increase. The optimization of porosity was reconfirmed by a control experiment on freshly prepared nSTFs with different porosities, which were spin coated with 0.6% rhodamine123 in ethanol (with a RPM 3000). The recorded surface enhanced fluorescence (SEF) spectra for these nSTF chips spin coated with rhodamine123 have been plotted in Fig. 4(b). It can be observed that a response similar to that observed in Fig. 4(a) is obtained, which reconfirms that 30% porosity has the optimum enhancement. The CO-nSTFs with the optimum porosity were used for the fabrication of the sensor chip.
2.1. Estimation of the enhancement factor
Because of the enhanced plasmonic field of the Ag nano-rods, the intrinsic fluorescence obtained from the CO-nSTFs is enhanced manifold as compared to simple CO configurations. The order of enhancement posed by the plasmonic character was estimated as follows. For the estimation of the enhancement, freshly prepared nSTF chip was incubated in 1% 4-ATP (wt. %) in ethanol for 2 hours to form a self-assembled monolayer of 4-ATP on the metal surface. Afterwards, the chip was washed thoroughly with ethanol and water and dried in a stream of nitrogen gas. The SEF spectra from the 4-ATP/nSTF chip and fluorescence spectra from a chunk of bulk 4-ATP were recorded using the setup mentioned in Fig. 1. The enhancement factor is given as:
where is the SEF intensity from the 4-ATP molecule adsorbed on the nSTF,is the fluorescence intensity from the bulk chunk of 4-ATP, is the number of adsorbed 4-ATP molecules contributing in the SEF signal and is the number of molecules from the chunk of 4-ATP which are contributing in the fluorescence signal. Let us assume that the area of the focused spot of the incident light is A. Then, , where dpen is the penetration depth of incident light in the solid 4-ATP sample and Nd4-ATP is the number density of the 4-ATP molecules in solid chunk. Utilizing the density of solid 4-ATP = 1.18 g/cc and molecular weight 125.19 g/mol, Nd4-ATP = 5.68 × 109 for an area of 1 μm2. Considering dpen = 100 μm2, NBulk = 5.68 × 1011. The corresponding fluorescence signal was recorded to be 759 counts (arb. units). The number of molecules adsorbed on the chip is given by , where is the concentration of the 4-ATP solution, is the column density (number of nano-columns per unit area), is the area of a single nano-column, and is the area of a single 4-ATP molecule. The factor is important when the concentration of the solution is smaller than that for the full coverage of surface. A nSTF chip of 5 × 5 mm2 dimensions and 30% porosity requires about 1.02 × 1015 4-ATP molecules to form a monolayer of it; with the area covered by a single 4-ATP molecule being 0.2 nm2. A volume of 50 μl of 1% (wt.%) solution of 4-ATP in ethanol (density = 0.789 g/cc) has 1.9 × 1018 molecules. Since the chip was incubated in 50 μl volume of this solution, it has sufficient no. of molecules for full monolayer coverage of the chip and hence, the factor . For a chip with 30% porosity, the number of 4-ATP molecules per μm2 on the nSTF is 4.08 × 107. The SEF signal recorded from this chip was 58 counts (arb. units). Hence, the total SEF enhancement per molecule was estimated to be 1.06 × 103, while that on the tip of nanorods was 1.45 × 104. Thus, the claimed order of fluorescence enhancement is rather under-estimation. Fluorescence enhancements of 3 orders of magnitude were only recently reported on a rather complicated geometry [44].3. HbA1c detection
3.1 Fabrication of the sensor chip
A schematic of the sensor chip fabrication is shown in Fig. 5. The surface of the CO-nSTF with 30% porosity was first immobilized by 4-Aminothiophenol (4-ATP). The CO-nSTFs were incubated in 1% 4-ATP solution in ethanol (wt%) for 4 hours to form a self-assembled monolayer (SAM) over the nSTF. The SAM formation occurs due to thiol binding and further, the –NH2 group on the other terminal of 4-ATP provides an opportunity for functionalization of BREs such as antibodies, bacteriophages, enzymes, etc. The 4-ATP/CO-nSTF surface was further immobilized by EDC-NHS modified anti-HbA1c antibody which is a key BRE for specific detection of HbA1c. Briefly, 200 μl solution of 0.2 mM concentration of EDC and that of 0.05 mM of NHS both prepared in 50 mM PBS, were mixed in equal volumes with antibody serum and then allowed to react for 2 hours at 4°C. The 4-ATP coated CO-nSTFs were then incubated in the EDC-NHS modified antibody serum for overnight at 4°C to form a SAM. EDC is a frequently used cross-linker of almost negligible length and is used for the activation of proteins or sensor surfaces for effective binding. EDC conjugates with any protein in two sequential steps: at first it modifies the carboxyl groups (-COOH) of the protein in an amine-reactive intermediate, called O-acylisourea, which intermediately reacts with the amine groups of the sensor surface/ protein to form a stable amide bond. The intermediate compound is quite unstable and is susceptible to hydrolysis as well. The instability of O-acylisourea leads to the regeneration of carboxyl groups and release of an N- unsubstituted urea and hence to low coupling efficiency. The role of NHS is to stabilize this intermediate by converting it into a considerably more stable amine-reactive NHS ester. This type of cross linking increases the coupling efficiency by 10-20 fold. The conjugation of the antibody with EDC-NHS allows the direct immobilization of the antibody on the 4-ATP coated CO-nSTF. The sensor surface was further incubated in 1mg/ml solution of BSA in 50 mM PBS buffer for 1 hour. The BSA works as an anti-fouling agent and prevents the possibility of any-non-specific binding on the sensor surface. This improves the specificity of the sensor chip towards the analyte.
After the fabrication, the sensor chip was stored at 4°C until it was used for sensing applications. Sample solutions of varying concentration of HbA1c ranging in the human physiological range (4-8% of Hb) were prepared in 50 mM PBS. About 10 μl of each sample solution was allowed to interact with the sensor chip for 2 minutes and then washed with PBS to remove any unbound HbA1c. Afterwards, the chip was blow dried with nitrogen and corresponding fluorescence image and spectrum were recorded. The sensor chip was regenerated for next use by washing the sensor chip with running 50 mM glycine buffer for3 minutes. Afterwards, the chip was washed with PBS and water and then dried to use for the next sample solution.
Figure 6(a) shows the intrinsic fluorescence spectra obtained from the sensor chip for different concentrations of HbA1c. It can be observed that for every concentration, the characteristic intrinsic fluorescence spectrum similar to that from bare CO-nSTF was obtained. With an increase in the HbA1c concentration, a trend of decrease in the fluorescence intensity was observed. As stated before, the concentration range was calculated to be equal to 4-8% of the physiological range of Hb. The reason for decrease in fluorescence intensity is the blocking of the fluorescence sites by the addition of HbA1c molecules on the sensor chip. The capability of the regeneration of the sensor chip adds the advantage of reuse and we could use the chip for few days during a period of three months. Hence, the chip has a large shelf life as well. The repeatability of the response was checked by repeating the fabrication of new chips and recording the fluorescence images and spectra for three times and a reasonably good repeatability was obtained. Negative control experiments on Hb were performed to ensure the specificity of the present sensor. The sample solutions of varying concentrations of Hb, similar to that of HbA1c were prepared in 50 mM PBS and the corresponding fluorescence images and spectra were recorded. Figure 6(b) presents the intrinsic fluorescence spectra for varying concentrations of Hb. The sensor chip was interacted with Hb sample solution for 2 minutes and then washed subsequently with PBS and blow dried with nitrogen gas before the recording of the fluorescence signal. After that, the sensor chip was regenerated and used again, as mentioned previously. It can be observed that there is no change in fluorescence intensity with increase in the Hb concentration. Even though we have not shown the fluorescence images, they were recorded as well for all the sample solutions. In Fig. 7, the response curve of the sensor is presented with varying concentrations of HbA1c and Hb. In this graph, we have plotted the change in the maximum fluorescence intensity on the left Y-axis and gray counts obtained from the recorded fluorescence images on the right Y-axis with the change in HbA1c/Hb concentration. The maximum fluorescence intensity counts were obtained from the Figs. 5(a) and 5(b), while the gray counts were obtained by processing the fluorescence images with ImageJ freeware from National Institute of Health (NIH) [45]. It can be observed that both the fluorescence intensity and the gray counts decrease with the increase in the HbA1c concentration, while they remain almost constant for increase in Hb concentration. It means that the sensor is specific to HbA1c only. Further, the error bars were added by considering the repeatability of the response and the accuracies of the pipette, timer and the spectrometer. It can be seen that reasonable repeatability was obtained. The beauty of the CO-nSTFs is that they operate on the principle of fluorescence based detection while being employed for label free detection.
Fig. 6 Enhanced intrinsic fluorescence spectra for varying concentrations of (a) HbA1c- and, (b) Hb- on the CO-nSTF sensor chip.
Fig. 7 Response curves obtained from the intrinsic fluorescence images and spectra of the sensor chip for varying concentrations of HbA1c and Hb.
4. Conclusions
Novel CO-nSTFs of Ag with enhanced intrinsic fluorescence have been reported. The sources of fluorescence were attributed to formation of Ag2O and residual carbon on the outer layer of the silver film which was supported by the XPS measurements. The structure was optimized with respect to porosity and it was found that CO-nSTFs with 30% porosity have the optimum fluorescence. The enhancement factor for fluorescence was estimated to be around 103 per molecule. Label free detection of HbA1c in human physiological range was demonstrated. Specificity of the sensor was established by control experiments on Hb. The present CO-nSTFs can be useful in various optical devices such as optical memory, label free ELISA, label free fluorescence biosensors, and other fluorescence techniques without a label.
5. Experimental section
5.1. Fabrication of the CO-nSTF
When the vapor is incident with a large angle (> 70°) with repect to the surface normal, nanorods are usually produced. This kind of physical vapor deposition is referred to as oblique angle deposition (OAD). As an extension of OAD technique, glancing angle deposition (GLAD) is a combination of OAD and an azimuthal rotation of the substrate.
The Ag nSTFs were grown on Si substrate by glancing angle deposition (GLAD) technique using ion beam sputtering [46]. The growth of these Ag columns utilizes the mechanisms of atomic shadowing and diffusion of deposited atoms (adatoms) at higher temperatures. A schematic of the GLAD setup and growth mechanism is shown in Figs. 8(a) and 8(b).
Fig. 8 Schematic of - (a) GLAD setup, (b) nano-columns formation; (c) SEM images of nSTFs of 30%porosity and 300 nm height.
In the used GLAD setup, the substrate is kept at very large angle between 70° and 85° to the incoming metal vapor. In the beginning stage of the deposition, self-organized nucleation sites are formed on the substrate. These nucleation sites play the key role in shadowing mechanism during the subsequent deposition and lead to highly directional growth of elongated nanostructures. By controlling the rate and time of deposition, deposition angle, rotation frequency, and temperature of the substrate, the topography, height and shape of nano-sculptured thin films (nSTF) can be designed [46,47]. Ag nSTFs of 300 nm height and different porosities ranging from 0 to 60% on Si (100) substrates were prepared by a rotation frequency of about 100 rotations per minute. These values were chosen because it was found to be around the optimum for the maximum SERS enhancement from our previous studies [48]. The Ag CO-nSTFs were obtained as a result of simple exposure of the as prepared Ag nSTFs in air. Figure 8(c) shows the scanning electron microscopic (SEM) images of the CO-nSTFs with 30% porosity.
5.2. Reagents
The 4-Aminothiophenol (4-ATP), N-hydroxysuccinimide (NHS), N-ethyl-N-(3-dimethylaminopropyl carbodimide) (EDC), rhodamine123, phosphate buffer saline (PBS), hemoglobin and glycine were purchased from Sigma Aldrich. Ethanol (98.99% pure) was purchased from Bio Lab Ltd., Israel. Anti- HbA1c antibody and HbA1c were purchased from antibodies-online GmbH, Germany. All these chemicals were used without any further purification. Water used for making buffers was taken from a Millipore® system.
Funding
Campus for Research Excellence and Technological Enterprise (CREATE) project NEW; the National Research Foundation of Singapore; Deutsche Forschungsgemeinschaft (DFG) trilateral programme (Germany-Israel-Palestinian Authority); Graduate school BuildMoNa (University Leipzig, Germany); the German Excellence Initiative of the DFG.
Acknowledgments
This research is conducted partially by NTU-HUJ-BGU Nanomaterials for Energy and Water Management Programme under the Campus for Research Excellence and Technological Enterprise (CREATE), that is supported by the National Research Foundation, Prime Minister’s Office, Singapore. The support by the Deutsche Forschungsgemeinschaft (DFG) within the trilateral programme (Germany-Israel-Palestinian Authority) and the Graduate school BuildMoNa (University Leipzig, Germany) funded within the German Excellence Initiative of the DFG are also appreciated. Sachin K Srivastava thanks the Council of Higher Education of the Government of the State of Israel for PBC post-doctoral fellowship.
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