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Studies of functional nucleic acids modified light addressable potentiometric sensors: XPS, biochemical assay and simulation Yunfang Jia, and Fang Li Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018
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Analytical Chemistry
Studies of functional nucleic acids modified light addressable potentiometric sensors: XPS, biochemical assay and simulation Yunfang Jia*, Fang Li College of Electronic Information and Optic Engineering, Nankai University, Weijin Road, Nankai District 300071, China. ABSTRACT: Functional nucleic acids (FNAs) are promising sensing elements, extensive interests are excited to integrate FNAs with transducers for biochemical assays. However, efforts for FNAs modified light-addressable-potentiometric-sensor (FNA-LAPS) are rarely reported. LAPS is a versatile transducer with electrolyte-insulator-semiconductor (EIS) structure, can respond almost any surface electronic deviations. Herein, organized studies for FNA-LAPS including experiments, theoretical derivations and MEDICI (Synopsys®) simulations, are presented using Pb2+-DNAzyme GR-5 and Ag+-aptamer as proof-of-concepts, which are typical FNAs with distinctive sensing strategies. Firstly, the on-LAPS occurrences of FNAs and their particular sensing actions are evidenced by tracking their X-ray photoelectron spectroscopy (XPS) core spectra of N1s, P2p, C1s, Ag, etc.. Then, applications of FNA-LAPS are executed by a home-made and mobile-phone controlled system, the limit-of-detection is 0.01 ppb, sensitivities are 2.86 (Pb2+) and 1.53 (Ag+) mV/log10(ppb) (R2=0.98), respectively. Furthermore, a charge and resistor mechanism (C&R) is proposed to illustrate the measured LAPS responding for FNAs and their sensing behaviors (Pb2+-mediated cleavage and Ag+mediated folding), based on carefully analyzing basic LAPS' experimental data and MEDICI calculated distributions of build-in potentials, energy-bands, carriers, etc., at EIS micro-interface (semiconductor side). Finally, demonstrations for C&R based FNALAPS principle are provided by the use of MEDICI, as a means to bridge experiments and theoretical deductions. In general, a cross-study for FNA-LAPS is proposed including XPS characterization, biochemistry detection, theoretical analyzing and MEDICI simulation, it is believed their powerful combination would provide an ideal workstation for analytical chemistry applications, not only the traditional determinations but also facilitations for investigating FNAs' configurational transformations.
Functional nucleic acid (FNA) is an artificial biomolecules by organic synthesis, its brilliant nature in precisely replicating creatures and specificity to the target of choice attracted researchers to devote their efforts into this field.1-3 In the discipline of biochemistry sensors, FNAs are believed to be excellent candidates as the sensing elements, they can be integrated with almost all kinds of transducers. A host of works have been reported to realize FNAs switched sensors,4,5 amplification strategies,6-8 directly electric FNAs sensing platforms9-12 and efficient detection system.13-16 The reported numerous works have been systematically classified in the earlier and very recent reviews,17,18 its crucial ability in specifically distinguishing the given target earns it the privilege of being called a new generation of biosensor, named as FNA sensors including genosensor, aptasensor and DNAzyme sensor.18 Benefited by the novel sensing strategies, the detections for metal ions have an opportunity to escape from the shackles of the heavy and costly equipments,19 they provide effective tools for realizing the in-field and portable environmental monitoring,20-22 and even the trace detection (limit of detection, LOD 26 zM) metal ions can be realized.23 Meanwhile, light addressable potentiometric sensor (LAPS) as a typical field-effect transducer with electrolyte-insulatorsemiconductor (EIS) structure,24 its smooth and insulating surface is favorable for micro-fluidic device,25 chemical imaging,26 accommodating versatile biochemical assays for biomarkers27 and cancer cells26, etc.. However, to our best knowledge, only three reports are found, they belong to two of three FNA sensors 18, and named as: (1) genoLAPS, which is modified by single strand DNA (ssDNA) to monitor DNA
hybridization;28-30 (2) aptaLAPS, which is modified by aptamer to realize Hg+ detection.31 The work for DNAzyme type LAPS (DNAzyme-LAPS) is missing. Moreover, in-depth understanding for the intrinsic mechanisms of FNA-LAPS experimental results are still in necessary, since the current surface charge (SC) sensing mechanisms28-31 can not explain why LAPS output signals (Vout or Iout ) can respond the erecting FNAs reaction,31 and our measured changing of Vout (∆Vout) caused by long FNAs is smaller than short ones. Though the researches about charge transfer (CT) studies of FNAs may provide an answer, which indicate longer FNAs chains tend to have bigger resistances (RCT).32 That is to say, FNAs folding (like T-Hg-T or C-Ag-C) induced variations (in chain length and cross section) can cause its resistor changing (∆RCT). Whether it can be transplanted to explain LAPS' ∆Vout, it still needs to be argued. Besides, some of the electrochemical impedance spectroscopy (EIS) studies are also in contrary, for the similar T-Hg-T hairpin structure, both lowered16 and increased11,23 RCT have been reported. Herein, studies for FNA-LAPS are carried out by the use of Pb2+-DNAzyme GR-533 and Ag+-aptamer6 as proof-ofconcepts, which are named as FNA-Pb and FNA-Ag, respectively, because of their typical and distinctive sensing procedures and ecological importance.34,35 Experiments are provided to evidence the occurrences of FNAs modification and their reactions on LAPS, including core X-ray photoelectron spectroscopy (XPS) (N1s, P2p, C1s, etc.), chemical assays by proposed FNA-LAPS and the home-made and mobile-phone controlled LAPS measuring system. Based
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Scheme 1. Experimental protocols for FNA-LAPS.
(A) FNA-LAPS surface modification, immobilization of DNAzyme FNA-Pb and aptamer FNA-Ag by covalent binding, Pb2+-mediated cleavage effect and Ag+-mediated hairpin structure. (B) LAPS testing unit, the reference electrode (R.E.) is Ag/AgCl, the counter electrode (C.E.) is Pt, LAPS is the work electrode (W. E.). Illumination from backside with wavelength 850 nm, power 30 mW. (C) FNA-LAPS model.
on XPS proofed on-LAPS FNAs sensing behaviors and measured LAPS' responding for them, sensing mechanisms of FNA-LAPS are deduced and simulated by MEDICI (Synopsys®) which is a popular device simulator, with the aims of profiling a dynamic-image for FNAs configurational transformations by the use of LAPS electronic signals.
EXPERIMENTAL SECTION Materials. FNAs are from Sangon Inc. (Shanghai, China), their structures are in Figure 1. In which, the sequences for FNA-Pb and FNA-Ag, are: 5'-CATCATCTCTTGCCGCCGG ATGAAGATAGTGAGAAACTCACTATrAGGAAGAGAT GATG-(CH2)6-NH2-3' and 5'-NH2-(CH2)6-CCCTTCCCTTCC CTTTTTTCCCAACCCAACCC-3', respectively. FNA-Pb is in double strand FNAs (dsFNAs) state, two complementary parts (underlines and double underlines parts) folded at the turning position of "AAA"; when Pb2+ exists FNA-Pb is cut at "rA", the left part curved naturally, as illustrated in the left column of Scheme 1(A).33 FNA-Ag is in single strand FNAs (ssFNAs) state, six thymine nucleotides ("TTTTTT") are the bending sites to form a hairpin structure when Ag+ is present, as depicted in right column of Scheme 1(A).6 Chemicals used in modification procedure are 3-aminopropyltriethoxysilane (APTES) (Sigma-Aldrich Co. Ltd, China) and glutaraldehyde (GA) (Alfa Aesar Co. Ltd.). Sample solutions are Pb2+, Ag+, Na+, Mg2+, Ca2+, Ni2+, Fe3+, Co2+, Mn2+, Cr3+, Cu2+ , Hg2+ and
Zn2+, in tris(hydroxymethyl) buffer (tris-buffer) solutions. Other chemicals are all analytic reagents, without further purification. The main materials for fabricating LAPS are ptype Si chips, aluminum, silicon nitride, silicon oxide; for building its measuring system are Bluetooth module HC-06 (Chuanglianfa Technology Co. Ltd., Shenzhen, China), Field Programmable Gate Array (FPGA) chip EP3C10E144C8 (Altera Corporation, USA), digital signal processing (DSP) chip TMS320F28335, Analog to Digital Converter (ADC) ADS7841 and Digital to Analog Converter (DAC) DAC7554 (Texas Instruments, USA). LAPS modifications. (1)APTES&GA: Piranha solution cleaned LAPS are incubated with APTES solution with the volume ratio (V/V) of 10% (pH=7.4), at 50℃ for 2h; then incubate them with GA solution 2.5% (V/V) at room temperature for 1h. (2) Pb-LAPS and Ag-LAPS: Incubate APTES&GA modified LAPS in FNA-Pb's or FNA-Ag's phosphate buffer solution (PBS, pH=7.4) , respectively, in the thermostatic oscillator (60 rpm, 37℃) for 4 hours. Testings on LAPS system are performed after each of the modifications, as shown by the blue arrows in Scheme 1, further sample tests are: (1) target ion's measuring. Incubate Pb-LAPS and Ag-LAPS with its target ion's tris-buffer solutions, respectively. After rinsing with DIW, examine its Vout. Incubation times and pH are examined from 15 to 40 min and pH=6.4 to 9.5. (2) Selectivity test. Incubate Pb-LAPS and Ag-LAPS with metal ions' solutions (tris-buffer and tap-water, 20ppb) respectively. All the measurements are executed by the same three-electrode LAPS (3E-LAPS) testing unit and the controlling system (in Figure S2). LAPS detection setup consists of two parts, shown in Figure S2. (A) is the mobile phone controlling terminal based on Android OS. (B) is the detection terminal including Bluetooth unit (a) to transfer user's commands from (A) to (B), and LAPS' data from (B) to (A), Vout signal processing module (b) to de-noise, amplify and convert the raw analog signal to digital data under controlled by DSP, control modules (c) and (d) to provide electronic and illumination power for LAPS testing unit (e), which is similar to Scheme 1 (B). FNA-LAPS Model. Scheme 1(C) is FNA-LAPS equivalent model, which is based on LAPS-pH model36 and EIS theory37 with two up-dates: (1) MEDICI "PHOTOGEN" procedure is used to simulate the dynamic balance of carrier's photogeneration and recombination, instead of the static current source38; (2) Electrolyte-insulator (EI) interface of LAPS is modeled by a variable resistor (RP) according to CT studies of NAs,32 instead of Hemholtz capacitor and Gouy-Chapman capacitor which are used to model H3O+-layer in electronic double layer (EDL).38 Because APTES&GA modification transforms the hydrophilic surface to hydrophobic. Other components COX, CD and RD are the equivalent components at IS interface, i.e. capacitor of silicon oxide layer, capacitor and resistor of depletion layer. XPS experiments are performed by Axis Ultra DLD (Kratos Analytical Ltd., UK). Seven XPS samples are: (1) BLANK, cleaned LAPS by piranha solution; (2) APTES&GA, after being modified by APTES and GA; (3) FNA-Pb, being immobilized by FNA-Pb; (4) and (5) Pb-ur and Pb-r, means not-rinsed and rinsed FNA-Pb chips after being incubated with Pb2+ solutions, respectively; (6) FNA-Ag, being immobilized by FNA-Ag; (7) CAgC, after being incubated with Ag+ solution and rinsed. Their core spectra are decomposed with ® the use of CasaXPS .
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Analytical Chemistry
RESULTS AND DISCUSSION
Figure 1. XPS characterizations for LAPS' surfaces. N1s core spectra of samples: (A) BLANK and APTES&GA; (B) FNA-Pb and Pb-r; (C) FNA-Ag and CAgC. The cross-comparisons of (A), (B) and (C) are provided in Figure S3(B). (D) P2p core spectra of samples FNA-Pb, Pb-ur, Pb-r, FNA-Ag, CAgC and APTES&GA. P element in different states are depicted in the insert-map. P1: paired-state in FNA-Pb and C-Ag-C structure; P2: unpaired-state, like P2_left, P2_cut and P3 in FNA-Ag.
XPS characterizations. According to the XPS studies of DNA39,40 and silicon nitride (Si3N4)41, N1s core spectra are used an indicator to trace the modification effects, FNAs immobilization and its reactions, and compared in Figure 1(A) to (C). N1s split peaks at 397.1-397.45 and 401.7±0.15 eV are found in them, they are assigned to N-Si3 and oxidized N (O-N) groups, in agreement with the Si3N4 layer used in LAPS.41 Peaks @398.6 eV are attributed to the neutral imine nitrogen groups,42 may be induced during preparing Si3N4 and APTES&GA modification. The extracted data indicate that NSi3 groups are reduced from 79% to 68%, imine and oxidized groups are enhanced from 20.0% to 27.2% and 1.5% to 4.4%, by organic modifications of APTES and GA. In Figure 1(B), peaks of @399.05, @400.3 and @400.3 are found in FNA-Pb and Pb-r, correspond to N-groups in NAs: imine (N1, ~399.0±0.1 eV), imine N acting as donor in hydrogen bonding (N2, ~399.1±0.1 eV), amine N acting as electron acceptor in hydrogen bonding (N3, ~400.0±0.1 eV) and amine N (N4, ~400.8±0.1 eV).40 In Figure 1(B) and (C), peaks of @398.7 and @399.05 belong to N1 or N2, peaks of @400.3 and @400.4 belong to N3 or N4. N1s core spectra of Pb-ur and quantitative comparing of three samples (FNA-Pb, Pb-ur and Pb-r) are provided in Figure S3, Pb2+-mediated cleavage effect is evidenced by tracing the extracted data of them: lowered N3 from FNA-Pb (51.37%) to Pb-ur (34.43%), increased unpaired ones from FNA-Pb (34.1%) to Pb-ur (38.08%); then it is rinsed off, evidenced by the lowered N3 from Pb-ur (34.43%) to
Pb-r (24.65%), and increased unpaired ones from Pb-ur (38.08%) to Pb-r (57.07%). In FNA-Ag samples, there is similar peak of @399.05 belongs to N1, but changed in contrary trail: in (C) it is lowered from 26.76% to 20.44%; in (B) it is increased from 34.1% (@398.7 eV) to 57.15% ( @399.05 eV). It is coincident
with their sensing principles: in the presence of Ag+, the ssFNAs (FNA-Ag) are fold, unpaired N groups are turned to paired ones; in the presence of Pb2+, the dsFNAs (FNA-Pb) are cut, paired N groups are turned to unpaired ones. There is a deviation between peaks @400.4 or @400.3 in (B) and 399.9 in (C). It may be caused by differences between C-Ag-C and normal base pairs. Phosphorous component can also be regarded as an index to proof the addition and subtraction of FNAs. In Figure 2(D), all the measured XPS data are treated by the same peak-splitting method, but only Pb-ur and C-Ag-C could be separated, P2p peaks are classified into two groups: around 133.32 and 134.0±0.1eV. They agree with the reported P2p core spectra of DNA molecules, which are centered at 133.4±0.1 eV40 and 133.8 eV.39 Peaks of @132.32 and @132.31 relates to P in dsFNAs, like P1 and P4 in the insert-map of Figure 1(D); peaks of @134.12, @133.98, @133.91 and @134.0 belong to unpaired ones, like P2 and P3. Then, the story for FNAs and their bio-behavior is profiled: after FNAs immobilization, P2p in dsFNAs (@133.32) and ssFNAs (@134.0) are found in FNA-Pb and FNA-Ag; after FNA-Ag being incubated with
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Figure 2. Basic photoelectronic features of LAPS and simulated distributions of potential, energy level, carriers. (A) The measured Vout at different VRE and Fmod. The symbols and error bars are mean values and standard deviations of the measured data (n=30). (B) Simulated distributions of build-in potential (ψ) at different VRE and currents distribution. (C) Diagram for ψ. The calculated energy bands include the conductive band (EC), valent band (EV) and the central energy (Ei). ψ is defined as (Ei-EF)/e, EF is the Fermi energy level (EF). The electrons (CE) and holes (CH) distributions, and photo-generated carriers (Cphoto) when VRE=0 and 0.8 V. (D) The electronic field distributions along "Y" and in the Si wafer (the insert-map). The positon of "Y" is the defined in Scheme 1(A).
Ag+ solution, C-Ag-C hair pin structure can be formed and characterized by
[email protected]; after incubation of FNAPb with Pb2+ solution, the cleavage effect can be identified by peaks
[email protected] and
[email protected], and the lost of peak
[email protected] from Pb-r indicates cut parts are washed away.
Core spectra of C1s, Ag3d and wide spectra provided in the Figure S3, they are in agreements with the reported data.39,43 We failed to find Pb in XPS, probably because it cannot be absorbed on LAPS flat and smooth surface . Basic photoelectronic features of LAPS. LAPS basic photoelectronic features are examined by phone-controlled system at varied VRE (0−1 V) and illumination modulation frequence (Fmod, 1000−4000 Hz) before FNAs immobilizations, shown in Figure 2(A). It is indicated that Vout is enhanced by the increased VRE. Though qualitative explanation has been provided for this observations,30-32,38 we still want to have an intrinsic view in LAPS, so that a clear computable LAPS principle could be formed, it will promote LAPS developing in a broader discipline. This work has not been reported up-tonow, and will be accomplished at here, for the first time.
The potential and current distributions calculated by MEDICI are shown in Figure 2(B), in which "Distance" is the position along "Y" direction, shown as the blue arrow in Scheme 1(A). When VRE is increased from 0 to 1 V, there are two parts at IS interface (Y=0−0.15 µm): (1) The sloping part from Y= 0−0.15 µm with the increased slopes and width when VRE is enhanced. (2) The flat part Y > 0.15 µm, only a small vibration in it. Meanwhile, enhanced current are also obtained, as shown in the inserted-map. The current distribution in 3Dmesh indicates that current is focused at the center area, that is the sensing and irradiated area as depicted in Scheme 1(B). These calculations indicate increased potential can accelerate carrier's movement and result in increased Vout. It needs to say the carriers and potentials in LAPS are different from in traditional electrochemical electrodes. Firstly, the potential is the build-in potential (ψ), formed by electric field effect, as illustrated in Figure 2(C). The initial distributions (solid lines) of electrons (CE) and holes (CH) are changed when VRE is changed from 0 to 0.8 V (as an example). Holes are expelled from and electrons are attracted to the IS interface. It contributes to the deviations of CE and CH
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Analytical Chemistry (bending dash lines). This variation is centered at Y< 0.2 µm, named as hole-depletion or space charge region. The energy bands in these areas are calculated and plotted, in which there are conductive band (EC), valent band (EV), the central energy level (Ei) and Fermi energy level (EF). ψ is defined by the band bending as: ψ=(Ei-EF)/e. The influences of VRE on CH, CE and ψ described by MEDICI simulations are: more holes will be evicted from IS interface by bigger VRE, thereby enlarged the space charge region, enhanced the band bending and ψ are induced. Secondly, carriers who make contribution to currents are photo-generated carriers. their concentration named as Cphoto is achieved by MEDICI shown in Figure 2(C), it agrees with back illumination method. LAPS' dependences on VRE and Fmod are also simulated and provided in Figure S4(C): no Vout can be measured, if no light or no VRE. Even at fixed VRE, the values of Vout are still altered by illumination conditions.32 Furthermore, simulated electronic field distributions are shown in Figure 2(D). Two peaks in the 3D-mesh map of wide-scale distribution correspond to the position of electrodes as depicted in Figure S4(A). There is also a nonzero electric field at IS interface, marked by a circle. Focused calculations for it indicate its intensity is enhanced by the increased VRE. This enhanced electronic field will boost the motion of photogenerated carriers which results in the increased current density shown in the insert map of Figure 2(B), and result in an increased Vout. Another parameter in Figure 2(A) is Fmod, it is used to modulate the period of illumination. At lower Fmod, more photo-carriers can be generated in one period, their separations by space-charge-region are also prolonged, which is expressed as prolonged current's period in Figure S4(D). This procedure can be described as a charging behavior to the hole-depletion layer, its equivalent component is CD as shown in Scheme 1(C). For same LAPS, CD is controlled by VRE, its alternating current (AC) impedance is in inverse proportional to Fmod ( 1 2πF C ). So, at fixed VRE, Vout is decreased with the mod
D
increasing of Fmod. In summary, LAPS basic principle is outlined as: (1) increased VRE will enhance space-charge-region in both width and intensity, then more photo-generated carriers can be separated in a faster way, therefore a bigger Vout can be measured; (2) at fixed VRE, the amount of separated photocarriers can increased by lower Fmod, then converted electronic signal Vout is enhanced. What is more, the slopes of the fitted lines in Figure 2(A) are also slightly varied with the lowering of Fmod. This phenomena can also be clarified by photocarriers motion mentioned above. That is: at lower Fmod, when there is a micro-changing of VRE (∆VRE), there will be enough time for photo-generated carriers to reflect ∆VRE in ∆Vout. Accordingly, in the following experiments, the working conditions is selected as Fmod =1000 Hz and VRE=0.8 V. LAPS responding for FNA immobilization. After being incubated with FNA-Pb and FNA-Ag in different concentrations (0.1−4 µM), Vout is measured and shown in Figure 3(A), named as Pb-LAPS and Ag-LAPS, respectively. There are similar tendencies for them, that is: Vout is lowered in low concentration of 0.1-1µM, then saturated at >1 µM. It can be explained solely by SC theory28-31 or RCT theory32, which is: grafted FNAs bring more negative charges or bring extra resistor (RP) on LAPS, ψ, electronic field and current are weaked; "saturated" means LAPS responding to FNAs is
Figure 3. LAPS responding for FNAs immobilization. (A) Measured Vout (symbols) of LAPS after FNAs immobilizations, named as Pb-LAPS and Ag-LAPS. The smoothed lines are obtained by OriginPro 8.1 SR3 with the method of SavitzkyGolay. The working conditions for these measurements are fixed at VRE=0.8 V and Fmod=1000 Hz. (B) Influences of VRE on FNAs immobilized LAPS, Fmod is fixed at 1000 Hz. The symbols and error bars are the mean values and standard deviations of the measured data (n=30).
limited by its capacity to accommodate FNAs. But, why Vout of Pb-LAPS (59 bases) are higher than AgLAPS (31 bases)? To make it clear, Vout-VRE features of PbLAPS and Ag-LAPS are examined and compared with LAPS before FNAs, named as APTES&GA, in Figure 3(B). It indicates, with binding of FNAs, RP are increased, the decreasing slopes correspond to it. Meanwhile, according to AC impedance 1 2πFmod C EI , CEI is the capacitor at EIS interface, like Cox and CD in Scheme 1(C). There is a capacitance-like effect, because all conditions (VRE, illumination power and Fmod) are fixed; and CEI,FNA-Ag
