DNA Templated Silver Nanoclusters for Fluorescence Based Detection of PfLDH
6.2 Experimental approaches .1 Chemicals
DNA probes were commercially synthesized by Bioserve, India and obtained in a desalted lyophilized condition. DNA probes longer than 30 mer were HPLC purified. All DNA probes were reconstituted in autoclaved MQ water (18.2 MΩ). Before using in experiments, all DNA probes were heated at 90 °C for 5 min, followed by cooling in ice for 2-3 min. AgNO3 (99.9 %) and 3-morpholinopropane 1-sulfonic acid (MOPS) were purchased from SRL, India. NaBH4(98 %), boric acid, HEPES, NaH2PO4, Na2HPO4, NaCl, acetic acid, sodium acetate, NADH, NAD+and Tris were purchased from Himedia, India.
Alcohol dehydrogenase (ADH) (cat no A7011) was obtained from Sigma. All reagents used were of analytical grade.
6.2.2 Preparation of AgNC
A total 6 µl of AgNO3(1.5 mM) was added to 15 µl of DNA probe (100 µM) and the volume was made up to 100 µl with Milli-Q (MQ) water. The mixture was stored in dark for 15 min after which 6 µl of NaBH4(1.5 mM) was added to it. The AgNCs were formed after 1.5-2 h at RT, in dark. The morphological characterization of AgNC was performed by Transmission Electron Microscopy (TEM) at an accelerating voltage of 200 kV (JEOL JEM, 2100).
6.2.3 Fluorescence, UV-visible, and circular dichroism (CD) spectroscopy
Fluorescence measurements were performed on a Tecan infinite M200 PRO, using black, flat, clear bottom fluorescence microplates (Thermo Fisher scientific) by diluting 5 µl of AgNC, in 95 µl of MQ water. Absorbance was measured on a UV/Vis spectrophotometer (Cary 100 Bio, Varian) between λ300 nm to λ800 nm using a quartz cuvette. For the measurements, 100 µl of AgNC were diluted with 900 µl of MQ water. The CD spectra were recorded on a Jasco J-815 spectropolarimeter calibrated with (+)-10- camphorsulfonic acid for optical rotation. The spectra were measured from λ350 nmto λ190 nm, using a 1 mm path length suprasil quartz cuvette at a scan rate of 50 nm.min-1, interval of 0.5 nm, time constant of 1 s, and taking an accumulation of 3 scans. For CD studies 10 µl of (100 µM) DNA probe was mixed with 2.7 M urea and the volume was made up to 200 µl using MQ water, obtaining a DNA: urea molar ration of 1.9. The mixture was stored for 20 min at RT before carrying out the CD experiment. For each probe analysis, a control experiment, with no added urea was also performed.
6.2.4 DNA PAGE
Native and denaturing gel electrophoresis were performed on 20 % polyacrylamide gels in a miniVE vertical electrophoresis system (Amersham) using TBE buffer (54 g of Tris base 27.5 g of boric acid, 20 ml of 0.5 M EDTA (pH 8.0)). For denaturing conditions the gel was run in the presence of 7 M urea, at RT. Native gel was run in a cold cabinet maintained at 4 °C. Both gels were run at 120 V, for 4 h, at RT. The gels were stained with SYBR gold dye (Invitrogen).
128 6.2.5 Effect of pH, temperature, denaturants, and time
The formation of the NCs was studied in buffers ranging in pH from 4 to 10. AgNC formation was carried out in 15 mM buffer solution for 2 h, at RT in the dark, followed by fluorescence measurement. To study the effect of temperature on AgNC formation, DNA probes were mixed with AgNO3and NaBH4at the required concentrations and the mixture was incubated at RT, 70 °C, and 90 °C water bath for 2 h, in the dark, followed by fluorescence measurements.
The interference in AgNC formation in the presence of DNA denaturants was studied. For this, AgNC nucleation was carried out in the presence of various denaturants followed by fluorescence measurement.
6.2.6 Interaction studies between AgNC and NAD+
In order to study the interaction of Sub 3-AgNC with NAD+ a series of solutions containing a fixed volume of AgNC (5μl) with increasing concentrations of NAD+(0-800 μM) was prepared. The binding process is described by the following equilibrium:
AgNC + n (NAD+) [AgNC-(NAD+)n] (1)
According to this equation, the AgNC binds with n equivalents of NAD+to form an AgNC- NAD+ complex. The fluorescence intensity at λ660 nm was used to calculate the binding constants (Ka) and number of binding sites (n) by using the Scatchard equation (Liang et al., 2008; Minet al., 2004).
log [(F0-F)/ F] = log Ka+ n log NAD+ (2)
In this equation, F0 is the initial fluorescence intensity prior to addition of NAD+, and F is the fluorescence intensity at a specific NAD+concentration. By titration of NAD+ and plotting log (F0- F)/F against log [NAD+], a linear curve was obtained with a slope of n. The y-intercept is equal to log Kaand provides the value of Ka(Kang et al.,2004; Minet al., 2004).
Both static and dynamic quenching processes are described by the Stern-Volmer equation:
F0/F = 1+ kqτ0[Q] = 1+ KD[Q] (3)
Where F0and F are the fluorescence intensities of the fluorophore in the absence and presence of quencher, [Q] is the concentration of the quencher, KD is the Stern-Volmer constant, kqis the bimolecular rate constant, andτ0is the lifetime of the fluorophore in the absence of quencher
Time resolved photoluminescence (TRPL) studies of the fluorescent AgNC and their complex with NAD+ were carried out on a picosecond time-resolved cum steady state luminescence spectrometer (Edinburgh Instruments, UK, Model FSP920) using an LED source, at an excitation ofλ599 nmand emission ofλ650 nm.
6.2.7 Interference studies
Specificity of AgNC for NAD+was assessed by monitoring the quenching of Sub 3- AgNC by various homologues of NAD+, namely NADP+, NADH and a synthetic cofactor APAD+. For this, 5 μl of Sub 3-AgNC was incubated with 5μl of a particular cofactor of 100 mM stock concentration, in a final volume of 100 μl, followed by fluorescence measurement. Interference to NAD+detection by Sub 3-AgNC was assessed by carrying out
130 the experiments in a background of monovalent or divalent salts. The effect of salts on plain Sub 3-AgNC was monitored by mixing 5 μl of Sub 3-AgNC with 8 μl of (100 mM) monovalent salts, or 10μl of (10 mM) divalent salts, in a final volume of 100μl. To validate the efficiency of NAD+mediated quenching of AgNC in the presence of salts, 1μl of (100 mM) NAD+was mixed with 5 μl of AgNC in the presence of 8 mM monovalent salt or 1 mM divalent salt, followed by fluorescence measurement. The applicability of the AgNC to measure NAD+ in serum samples was also investigated. Fresh human blood was collected from healthy human volunteer and centrifuged at 900 x g for 10 min to precipitate the RBCs.
100 μl of supernatant (serum) was collected, mixed with MQ to a final volume of 1 ml and stored at 4 °C for further use. To carry out the assay, we mixed 5 μl of AgNC, with 5μl of serum and varying NAD+concentrations, in a final volume of 100μl, followed by monitoring the fluorescence emission.
6.2.8 Turn-on, and turn-off fluorescence assay
For the AgNC-NAD+interaction based turn-on assay using ADH, 5 μl (1 mg.ml-1) ADH was mixed with 5μl (100 mM) NAD+, 5 μl (100 %) ethanol, 5μl (20 mM Tris-Cl, pH 8.8), 75 μl of MQ water, and allowed the enzyme catalysis for 5 min, followed by addition of 5 μl of AgNC. To carry out the turn-off fluorescence assay using PfLDH, 3.2 μl of (31 μM) PfLDH was mixed with 5μl (20 mM, pH 6) HEPES buffer, 10 μl (100 mM) NADH, 30 μl (100 mM) sodium pyruvate, and 46.8μl MQ water, and allowed the enzyme catalysis for 5 min, followed by addition of 5μl of AgNC. In each case a control experiment with no enzyme was also performed.