Nanostructured Memristor Sensor Mimics Acetylcholinesterase (ACHE) Active Sites In The Gorge For fM Detection Of Acetylcholine

E. T. Chen1*, J. Thornton2 C. Ngatchou1and S-H. Duh3 1Advanced Biomimetic Sensors, Inc., 13017 Wisteria Dr, #109, Germantown, MD 20874; 2 Bruker Nano, 19 Fortune Dr., Billerica, MA 01821; 3University of Maryland Medical System, 22 South Greene St, Baltimore,

MD 21201; * To whom it should be contacted: ellenchen@nanobiomimeticsensors.com.

ABSTRACT

Many diseases including cancer, diabetes, brain injury, epilepsy, Parkinson’s, autism and Alzheimer’s involve the dysregulation of acetylcholinesterase (ACHE) causing inappropriate production of the neurotransmitter acetylcholine (ACH). Providing a nonenzymatic detection device for ACH with rapid detection time and high sensitivity is paramount to avoid time consuming assays and protein interferences. We report new types of nanostructured memristor biomimetic ACHE sensors developed for detection of ACH without using nature enzymes and are reagent-free. Memristor sensor #1 was made for mimicking the normal active sites in the ACHE gorge and memristor sensor #2 was made for mimicking the mutated gorge with damaged eternal lining by knockdown of the 14 aromatic residues. Multiple polymers were cross linked and self-assembled on gold chips forming a “Flat-bridge/nanopore ACHE Gorge” for sensor #1 and a “Vertical bridged-nanopore ACHE gorge for sensor #2; and the i-V curves were observed of hysteresis loops. Results obtained by a Chronoamperometric (CA) method for quantitation of ACH revealed that sensor #1 has Detection of Limits (DOL) of ACH 9.7x10-16 M/cm2 over 10-15 to 10-6 M and an accuracy 101 ± 8% using spiked NIST SRM965A human sera against calibration curve. Sensor #2 was unable to detect ACH directly. Delta Slow-Wave-Sleeping (SWS) synapse firing from the two biomimetic neuron networks are compared. A 5.5-fold higher intensity for Sensor 1 was observed. Key Words: Nanobiomimetic Memristor Electrochemical Sensing; Acetylcholinesterase (ACHE) Gorge; Acetylcholine; Reagent-less; Slow-Wave-Sleeping (SWS); Nanobiomimetic Hyppocampal-Neocortical Models.

INTRODUCTION

Acetylcholinesterase (ACHE) is a very important

hydrolase. It is found in nerve and muscle, central and peripheral tissues, motor and sensory fibers, and cholinergic and noncholinergic fibers. Its primary function in high rate hydrolysis activity for terminating synaptic impulse transmission of neurotransmitter acetylcholine (ACH) [1-3] and ACHE’s non-cholinergic functions such as cellular proliferation are well known [4-5]; ACHE dysregulation promoted cancers were reported everywhere [6-8]. The

neurotransmitter ACH also plays an important role in spatial and contextual learning and memory [9-12]. During slow-wave sleep, however, declarative memory consolidation is particularly strong [13]. Pesticides (herbicides, fungicides, insecticides) widely used in the agriculture and industry, are neurotoxic compounds which irreversibly inhibit ACHE, resulting in the accumulation of the neurotransmitter ACH. Therefore, it is important to characterize the activity of ACHE through quantifying ACH with enhanced sensitivity and simplicity as reviewed in literature [14-15]. Improving the biosensor performance of ACH is challenged for unavoidable biofouling and nonspecific protein bounding caused interference by utilizing nature enzyme or coenzyme [16-19].

Biomimetic electron-relaying system, which not only mimics the active sites of the proteins, but also promotes direct bio-communication between the artificial active sites and the electrode by utilizing a nanostructured self-assembled membrane (SAM) films offering an attractive pathway to enhance the selectivity, sensitivity and environmental protectiveness. It was discovered that the structures of biomimetic enzyme sensor membranes played an important role in enabling selective detecting of toxins for being able to distinguish isomers and different types of cancers [16, 20-23]. However, a device with nature inspired dynamic dipole ACHE gorge [1-3] design would be more closely mimicking the environmental stimulation for catalyzing ACH. The attempt of this project is to develop a device that is able to mimic the gorge’s fast hydrolysis function without using antibody, ACHE and any tracer.

In review of the recent advances of the ACH sensor development, the most popular approaches are using ACHE nature enzyme or coenzyme fabricated on the substrate surface [14-15, 24-26]. The reported sensitivity reached 10 nM using this biosensor [14-15, 24-26]. As mentioned above, our approaches are to avoid using nature ACHE, but set up a biomimetic ACHE gorge providing a suitable conformational and functional “net” to attract the “fish” of ACH for a “direct meaningful bio-communication” without any reagent used, in order to be environmentally friendly and be able to have an order of magnitude higher sensitivity and portability. Further- more, a memristor type of sensor may closely mimic the nature of the ACHE in hippocampus and thalamic, because the ACH closely related to memory with a long, complex and

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chaotic but still living relationship [27-29], hence the new type of sensor may be revolutionizing the biosensor field.

Memristors and Memcapacitors are devices made of nanolayers that have the capability to mimic neuronal synapse with a characteristic of hysteresis loop in the i-V curve [30-34]. However, most of the memristors and the memcapacitors are made of metal oxide materials [29-34], that make mimicking the ACHE gorge’s function more difficult. The purpose of this research is to develop a memristor device that closely mimics the ACHE gorge with cross-linked nanostructured polymers without using metal oxide. A “Healthy Active Site ACHE Gorge” is defined as: Ser200-His400-Glu327(Catalytic Site (CAS)) mimicked by Polyethylene glycol diglycidyl ether (PEG)....imidazolyl-dimethyl-β-cyclodextrin (M-CD)...triacetyl-β-cyclodextrin (T-CD) and W84 mimicked by poly(4-vinylpyridine) (PVP); The 14 aromatic residues for gorge lining were mimicked by excess amount of o-NPA (1:500-1000 of T-CD/ o-nithophenyl acetate (o-NPA)). F330, Y121 were mimicked by o-NPA, and W279 was mimicked by PVP. By knock down all o-NPA out of the network, we define the second device as “Mutated Active Site ACHE Gorge” based on our hypothesis: Lacking of hydrophobic lining in the gorge might be the key issue caused diseases, because the nature of the ACHE gorge might be memristive.

EXPERIMENTAL

Fabrication of the Nanostructure Self-Assembling Membrane (SAM) Gold Memristor Chip The nanostructured biomimetic “Mutated ACHE Active Gorge” memrisor with the vertical bridged conformational/nanopore was freshly prepared and fabricated on gold chip. Polyethylene glycol diglycidyl ether (PEG), triacetyl-ß-cyclodextrin (T-CD), poly(4-vinylpyridine) (PVP) were purchased from Sigma. PVP was purified before use. The mono imidazolyl derivative dimethyl ß-cyclodextrin (mM-ß-DMCD) was generally synthesized according to the published procedures [35]. The appropriate amount of solutions of individual polymer and reagents were prepared [36]. The mixture solution was made up by mM-ß-DMCD, T-CD, PEG and PVP. The 16 channel gold electrode chips were purchased (Fisher Scientific). The mixture solution was injected onto the surface of the electrode, was incubated for 48 hrs at 37ºC [36] and all other clean procedures were followed by citation 36. This memristor was used as Sensor 2. The “Healthy Active ACHE Gorge” memrisor with the flat bridged conformation/nanopore was freshly prepared by adding appropriate amount of o-nitrophenyl acetate (o-NPA) into the above described mixture solution used for fabricating the vertical bridged memristor. All other procedures were

followed as cited in literature 36. This memristor was used as Sensor 1. Characterization of the Membrane

The morphology of the AU/SAM was characterized using an Atomic Force Microscope (AFM) (model Multimode 8 ScanAsyst, Bruker, PA). Data Collected in PeakForce Tapping Mode. Probes used were ScanAsyst-air probes (Bruker, PA). The silicon tips on silicon nitride cantilevers have 2-5 nm radius. The nominal spring constant 0.4N/m was used. Figure 1 illustrates the 3D vertical conformational bridge structure with “breathing nanopore” of the AFM images as sensor 2. Figure 2 illustrates the 3D flat conformational bridge structure with “breathing nanopore” of the AFM images of the Biomimetic Memristor 1.

Fig 1(L). 3D vertical conformational PDC bridge structure of

the AFM images of the Biomimetic memristor 2. Fig 2 (R) 3D horizontal conformational bridge structure of

the AFM images of the Biomimetic memristor 1.

Frequency Affects on Memristor’s Performance

Evaluations of frequency affect on memristors’ performance were conducted by Cyclic Voltammetric method (CV) in pH 7.40 saline solution at room temperature. For Sensor 1, 20 Hz to 500 Hz and 1 pM ACH was used with o-NPA. For Sensor 2, it had to be first used in the presence of 2 mM o-NPA and then 10 μM ACH can be measured, not requiring pM concentrations. Ratios of relative signal strength were used for comparison, i.e., j/cm2/unit ACH concentration, for Sensor 1 and 2, respectively, at the same frequency.

Quantitation of ACH Chronoamperometric method was used for quantitation of ACH (Acetylcholine chloride, Sigma) in pH 7.4 PBS at initial applied potential of -50 mV, then -200 mV for detection of ACH, the final concentration range over 10-15 to 10-6 M for sensor 1 at room temperature. Because Sensor 2 had difficulty detecting ACH, hence, we choose Sensor 2 to detect o-NPA instead, in the concentration range over 10-10 to 10-4M in the presence of 10 μM ACH using an electrochemical work station (Epsilon, BASi, IN) with the companied software package. Origin 9.0 was used for all statistic data analysis and figure plotting.

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Assessing Quality of Slow-Wave Sleeping (SWS)

The Double Step Chronopotentiometry (DSCPO) method was used for assessing quality of slow-wave-sleeping of the newly developed nanobiomimetic hyppocampal-neocortical neuronal model for evaluation of the SWS. It is well accepted fact reported by research groups that humans who lack quality SWS (Delta wave of 0.1-4 Hz) most likely will suffer with many illnesses, such as type 1 diabetes mellitus, seizures (Epilepsy), Alzheimer’s, Parkinson’s, brain injury and autism [13, 37-39]. Normal SWS waves have the highest amplitude compared with all other brain waves, because during the deep stage 3 or 4 sleeping, brain conducts consolidation of declarative memory through hippocampus to neocortical networking [13, 37-40]. We set up the frequency at 0.25 Hz with a current of ±10 μA and compared the signal strength between the “healthy active ACHE gorge neuron” and the “mutated ACHE gorge neuron”. The specimens used were NIST SRM965A human sera with blood glucose level 2 (70 mg/dL) with a data rate of 1 KHz without spiking ACH.

RESULTS AND DISCUSSIONS Characterization of the Memristors We have come to believe that the brain is a special memristor or memcapacitor type of device that is able to conduct complex learning and memory through chemical synapse and electric synapse of networking. The key structural difference between the two is the synapse gap. For electric synapse is one tenth of that of chemical synapse [41]. For Sensor 1, the 3D lattice between the flat bridge and the top rim of the surface of the pores are gaps of 40-56 nm; yet the Sensor 2 has gaps between 6-121 nm. Sensor 2 can become a hybridized memristor with bridges having 115 nm apart in height, and the Sensor 1 related to Sensor 2 having more characteristics of electric synapse than Sensor 2 as shown in Fig 3A (Sensor 1) and 3B (Sensor 2).

0.03 0.02 0.01 0.00 -0.01 -0.02 -0.03-4

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4Effect of frequncy on the Memristor 2 with vertical bridged/nanopore of Au/biomimetic mutated ACHE in the presence of 10 μM ACH and 2 mM o-NPA

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Fig 3A illustrates frequency affects on Memristor 1 with

typical bipolar nonlinear characteristics; Fig 3B shows Memristor 2’s typical bipolar linear behavior.

The Memristor 1 is 4580-fold more sensitive to

ACH than Memristor 2 at 1 μM ACH and 1 cm2 sensor area, indicating that a hydrophobic lining of the inner ACHE gorge is so critically important for learning and memory.

Quantitation of ACH Following are preliminary data demonstrating the performance characteristics of the ACH sensor 1 using a CA method. Sensor 1 was able to measure ACH from 10-15 to 10-6 M shown in Fig 4A and the current vs. concentration plot in Fig 4B was presented. Km is 0.24 nM by Lineweaver-Burk plot and Vmax is 0.61 nM/s. The DOL is 9.7x10-16 /cm2.

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Data: Data3_BModel: Exp1P1Equation: y = exp(x-A)Weighting: y No weighting Chi^2/DoF = 119.8256R^2 = 5.6417E-7 A -2.1021 ±0.5461

ACH Concentration (M)

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rent

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)

Au/"Healthy ACHE gorge" sensor 1 with 0.031cm2 detectsACH in PBS over fM to μM

Fig 4A illustrates CA curve profiles and 4B

shows the current vs. ACH concentration plot. The imprecision was 8.2 and 8.3% using NIST 965A human sera with and w/o spiked ACH with triplicate runs. Accuracy was 101% (LCI 85%; UCI 117%) of spiked NIST sera against the calibration curve at 1.2x10-7M ACH. Because the CA curves were sine waves, hence imprecision results were ±0.4-0.6% by averaging of each of the two groups. Mutated ACHE Gorge Needs a Hydrophobic Lining

It was observed that Sensor 2 lacking a hydrophobic lining in the gorge, was unable to sense ACH. Fig 5A and 5B illustrate the CA profiles and the calibration curve with a DOL of o-NPA 5.6 x10-13M/cm2 under 10 μM ACH.

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sensor 2 detects o-NPA in 0.01 mM ACH in pH 7.4 PBS,Y = -0.002 + 0.016X with Sy/x = 0.01, r=0.9998

n=15

J (A

/cm

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Fig 5B is the calibration plot.

Assessing Quality of Slow-Wave Sleeping (SWS) The DSCPO method was used for assessing quality of slow-wave sleeping of the newly developed nanobiomimetic hyppocampal-neocortical neuronal models. Fig 6 illustrates Sensor 1 has 5.5-fold higher peak intensity than Senor 2 in the Delta wave at 0.25 Hz, that indicates Sensor works perfectly at deep stage 4 sleep for memory consolidation; however Sensor 2 has the lowest wave intensity indicating loss of declarative memory and learning capability. It was noteworthy, that the healthy SWS has biphases, and the unhealthy SWS only has one phase with all peaks above zero

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100Frequncy effect on peak current of an 0.031cm2Au/"Flat bridge" nanopore ACH gorge model sensor detects1 pM ACH in PBS

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in which they are losing the membrane potential reversibility

[42-43]. 0 20 40 60 80 100 120

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A 0.031 cm2 Au Memristor/"Flat bridge-nanopore" "ACHE healthy Gorge" membrane with

an insulator/Pt in NIST Human sera with level 2 glucose 70 mg/dL. Electrodes were connected at 2250 at 0.25 Hz with ± 10 μA.(A); Au memristor/"verital-bridge-nanopore" mutatedACHE gorge at same experimental condition.(B)

Vot

age

(V)

Time (s)

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B

Fig 6. Compares Sensor 1 and 2 in the SWS waves at 0.25

Hz with Sensor 1 (black), Sensor 2 (red).

CONCLUSION Detection of ACH with accuracy and sensitivity in fM DOL under enzyme –free and reagent-free conditions was demonstrated and the Km value agreed with literature using nature ACHE [44]. Using human blood samples shown a 5.5-fold higher intensity in Delta SWS synapse by Sensor 1 compared with Sensor 2 indicate that enhancing the ACHE gorge hydrophobicity is necessary when its lining was damaged.

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