Ultrasensitive detection of Ebola matrix protein in a ...Ultrasensitive detection of Ebola matrix protein in a memristor mode Bergoi Ibarlucea1,2 ( ), Teuku Fawzul Akbar ... [11–13]

Ultrasensitive detection of Ebola matrix protein in a memristor mode

Bergoi Ibarlucea1,2 (), Teuku Fawzul Akbar1, Kihyun Kim3, Taiuk Rim3, Chang-Ki Baek3, Alon Ascoli4,

Ronald Tetzlaff4, Larysa Baraban1,2 (), and Gianaurelio Cuniberti1,2

1 Institute of Materials Science, Max Bergmann Center for Biomaterials, Technische Universität Dresden, Budapester Str. 27, Dresden

01069, Germany 2 Center for Advancing Electronics Dresden (CFAED), Technische Universität Dresden, Dresden 01069, Germany 3 Department of Creative IT Engineering, Pohang University of Science and Technology, Pohang 37673, Republic of Korea 4 Chair of Fundamentals of Electrical Engineering, Technische Universität Dresden, Mommsenstraße 12, Dresden 01069, Germany

Received: 16 April 2017

Revised: 2 June 2017

Accepted: 11 June 2017

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2017

KEYWORDS

memristor biosensor,

capacitance,

honeycomb nanowires,

silicon nanowire field

effect transistor,

VP40 matrix protein,

Ebola detection

ABSTRACT

We demonstrate the direct biosensing of the Ebola VP40 matrix protein, using a

memristor mode of a liquid-integrated nanodevice, based on a large array of

honeycomb-shaped silicon nanowires. To shed more light on the principle of

biodetection using memristors, we engineered the opening of the current-minima

voltage gap VGAP by involving the third gap-control electrode (gate voltage, VG)

into the system. The primary role of VG is to mimic the presence of the charged

species of the desired sign at the active area of the sensor. We further showed

the advantages of biodetection with an initially opened controlled gap (VGAP ≠ 0),

which allows the detection of the lowest concentrations of the biomolecules

carrying arbitrary positive or negative charges; this feature was not present in

previous configurations. We compared the bio-memristor performance, in terms

of its detection range and sensitivity, to that of the already-known field-effect

transistor (FET) mode by operating the same device. To our knowledge, this is

the first demonstration of Ebola matrix protein detection using a nanoscaled

electrical sensor.

1 Introduction

Nanosensors are currently attracting attention as pro-

mising tools for biotechnology, e.g., as a miniaturized

diagnostics laboratory; being able to sense pH [1–4]

close to the Nernst limit; and to detect biomolecules

[5–7], viruses [8], and cell activities [9] with high

sensitivity. Label-free, rapid, and ultrasensitive probing

of biologically relevant analytes is possible at a

nanoscale and at low cost thanks to devices that rely

on current or voltage changes that are altered by

charge redistribution on the active surface or in the

Nano Research

DOI 10.1007/s12274-017-1720-2

Address correspondence to Bergoi Ibarlucea, [email protected]; Larysa Baraban, [email protected]

| www.editorialmanager.com/nare/default.asp

2 Nano Res.

surrounding environment [10]. Among the multiple

classes of electronic sensors, devices measuring the

electrical impedance [11–13] and field-effect transistors

(FETs) [14–18] have been shown to provide the best

detection limits without sacrificing the possibility to be

miniaturized and implemented as wearable, flexible

devices [19]. FETs are the most-studied configuration,

since their introduction by Piet Bergveld [20] in 1970

and technological and conceptual upgrade using silicon

nanowires by Charles Lieber [4] approximately a decade

ago. Several works have shown the large number of

applications and possibilities that FETs offer [21–25].

However, they reveal an important drawback; the

sensitivity is lost when samples with a high ionic

concentration are measured, owing to shortening of the

Debye length—the distance within which the devices

are sensitive [26].

During previous years, strong efforts have been

dedicated to overcome the limitation of FETs in higher

ionic strength solutions or to gain enough sensitivity

to allow detection by simply diluting the sample.

Multiple strategies have involved surface chemical

modifications such as the incorporation of biomolecule-

permeable polymer layers [27] or gold nanoparticles

with antibody fragments [28]. Others, for example, rely

on new analytical procedures, such as the analysis of

antigen-dissociation kinetics [29], the analysis of high

frequency impedance signals [30, 31], and the operation

of the device by dual gates [32].

An alternative strategy is to explore the distinct

nanoelectronic configurations of the sensing elements

and measurement modes. In this context, after the

theoretical prediction of the existence of the memristor

in 1971 by Leon Chua [33] and the association of his

ideas with a fabricated device in 2008 by Strukov et al.

[34], studies of this new type of circuit element have

been conducted [35, 36], including Sandro Carrara’s

demonstration of their application as a new type of

biosensor [37]. Tzouvadaki et al. [38] demonstrated

that a non-zero concentration of charged biomarkers

opens up the semi-logarithmic output curves (drain

current–source-to-drain voltage I–VSD loci), under

alternating current (AC) excitation. In other words, the

perturbations caused by the biomarker presence on

the sensing surface (Fig. 1(a)) lead to the violation of

one of the memristor signatures, namely, the exhibition

of pinched (i.e., zero gap) current–voltage loci [39].

The appearance of a gap between the minimum current

peak values and the voltage width that separates

them (voltage minimum gap, VGAP, Fig. 1(b)) has been

associated with larger energy requirements for the

transport of charge carriers compared to that required

for the bare genuine memristor with no gap. The gap

depends on the amount and the charge sign of the

biomarkers. This leads to the high sensitivity, demons-

trated by the atto- and femtomolar detection of the

bioanalyte and confirmed by an AC analysis of the

binding molecules. Throughout the paper, these types

of experimental analyses are referred to as memristor

measurement mode. Although the high sensitivity is

proven, the technique shows a limitation marked by

the initial conditions of the gap and the charge sign

of the analyte to be detected. The charge sign of the

analyte (positive or negative) governs the processes

of the opening or closing of the VGAP. When the initial

measurements do not lead to the creation of a gap

(VGAP = 0), the technique is limited to detect analytes of

the charge type that will open it. Therefore, detection

of analytes of the opposite charge, e.g., analytes with

a gap-closing effect, will remain challenging. The

solution to this fundamental problem lies in establishing

an external control of the initial state (gap opening)

conditions.

Finally, the previously reported memristor biosensors

operate in dry conditions, which limits the range of

their potential applications in, for example, clinical

or point of care (POC) diagnostics, where the assays

are dominantly performed in liquid phase. Such dry

measurement formats aim to overcome the Debye

length limitation that decreases the screening length

capacity of these biosensors owing to the presence of

other ions in the sample [26]. Note that humidity

control is, however, crucial for the dry format, because

minor humidity perturbations would affect the width

of the gap [40]. In the context of POC applications,

typically applied out of the laboratory environment

with a minimum technological setup, measurements

in liquid phase are preferable. Very recently, such

measurements were demonstrated using a bare device

for pH sensing of buffer solutions [41], but biosensing

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

3 Nano Res.

in liquid samples is still unexplored.

Here, we attempt to solve the number of aforemen-

tioned shortcomings in the existing sensors measured

in memristor mode and present the first liquid

integrated bio-memristor based on a large array of

silicon-nanowire-based electrical devices. The pre-

sence of ionic species attached in proximity to the

semiconducting channel introduces a capacitive effect

that violates the memristor characteristics by modifying

the VGAP. The width change of this gap can be correlated

to the concentration of molecules in the sample. We

further engineered the gap opening process by involving

the third electrode, which acts as a gap-control

terminal (gate voltage, VG) that mimics the presence

of the surrounding charged species of the desired sign

(Figs. 1(a) and 1(b)). With the help of this electrode,

the creation of the VGAP in the output characteristics

can be done on purpose, to assure detection of analytes

of any charge sign at low concentrations. Finally, we

compared the bio-memristor performance, in terms

of its detection range and sensitivity, to that of the

already-known FET mode by operating the same

device. The demonstration is done for detection of

the Ebola VP40 matrix protein, which represents high

emerging relevance owing to its recent severe outbreaks

and high fatality rate [42]. A portable, miniaturized

biosensor that can be used in a non-specialized

laboratory or location is urgently needed for the low-

cost early detection of the disease, which would help

to prevent its spread. The most common diagnostic

methods for Ebola are the polymerase chain reaction

(PCR) and the enzyme-linked immunosorbent assay

(ELISA) [43]. To date, few alternative approaches have

been proposed, such as plasmonic [44] or surface

acoustic wave immunosensors [45] and fluorescence

DNA sensors based on nanoporous membrane systems

[46]. Despite the good results achieved, a purely

electrical biosensor would be preferable for the

Figure 1 Concept of the present work: (a) VP40 proteins from an Ebola virus bind to antibodies immobilized on a HC-FET and (b) theviolation of the memristor zero-crossing signature of the biosensor is measured. The opening or closing of the voltage minimum gap(VGAP = V1 – V0) when sweeping current to source-to-drain voltage VSD is plotted. (c) Device used in this work. An array of transistors isplaced surrounding a common reference electrode. The electrode design of the area marked in the white window is shown in (d).(e) Magnification of the same area on a fabricated device. S = source electrode, D = drain electrode, R = reference electrode. The insetin (e) presents a SEM image of the nanowire pattern. (f) Transfer characteristic of the HC-FET at fixed VSD = 0.1 V in dry conditions.Gate voltage VG is applied via a back gate. Inset shows output characteristics at three different VG. Dotted lines in both graphs representthe gate-to-drain current (leakage). (g) Transfer characteristic of the HC-FET at fixed VSD = 0.1 V in liquid conditions (deionized water).Upper inset shows output characteristics at three different VG. Dotted lines in both graphs represent the gate-to-drain current (leakage).Lower inset presents a TEM image of the nanowires’ cross-section.


4 Nano Res.

abovementioned reasons, leading to the implementation

of several optical setup-free nanosensors in a tiny chip.

We propose a direct, label-free, and rapid method for

the detection of VP40—a protein that contributes 40%

to the Ebola virus protein mass [47], making it the

ideal candidate as a biomarker for diagnosis. The effects

of the binding of this protein on surface-immobilized

antibodies were detected using the previously reported

high-performance honeycomb patterned nanowire FETs

(HC-FETs) [48, 49], showing remarkable sensitivity.

The gap-control terminal was used during the bio-

sensing, which, unlike in other memristor biosensors

[37, 38, 40], will allow us to set the desired initial

conditions for the detection of proteins of any charge

sign.

2 Results and discussion

2.1 Engineering of the voltage gap opening

A chip with an array of HC-FETs with a common

reference electrode was used in this study (Figs. 1(c)

and 1(d)); nanowires on this electrode were patterned

as a honeycomb structure (Fig. 1(e)) owing to their

proven higher sensitivity compared to that of linear

structures [49]. FETs with 32.5-nm-wide (average)

nanowire channels, lightly doped (1014 cm−2) with

phosphorus and patterned in a honeycomb shape

by electron beam lithography (EBL), were used for all

experiments (Fig. 1(e)). Source and drain regions were

highly doped with phosphorus. Source and drain

electrodes, as well as the VGAP-controlling reference

electrode for liquid gating, were fabricated via

deposition of stacked Ti and Ag layers and lift-off

processing. The devices were insulated with a 2-μm-

thick layer of SU-8 resist, leaving the nanowire area

and the reference electrode exposed to the environment.

The use of the EBL technique ensured good sensor-

to-sensor reproducibility on a wafer-scale fabrication.

More details on fabrication can be found in the

experimental section. The fabrication process resulted

in n-type FETs with a working range below VG = 30 V

in dry conditions (Fig. 1(f)) using the back gate,

whereas the VG needed for liquid conditions (Fig. 1(g))

using the reference electrode decreased by 10 times

owing to the thinner top oxide layer. Electrical mea-

surements were performed using a tip probe station

with PH150 micropositioners (SÜSS MicroTec, Garching

bei München, Germany) to connect the contact pads

and a source meter unit (2604B source meter unit,

Keithley Instruments GmbH, Germering, Germany).

The VGAP between the minimum current peaks in the

forward and backward sweep of VSD was calculated

using the following equation (Fig. 1(b))

VGAP = V1 – V0 (1)

where V1 and V0 are the current-minima voltages for the

two sweeping directions. For all the measurements,

the fastest scan rate allowed by the data acquisition

software (Matlab, Mathworks, Natick, MA, USA) was

applied (t = 10 s per sweep). As shown in Fig. S1 in the

Electronic Supplementary Material (ESM), at slower

speeds VG values that were more negative were needed

to open the VGAP, owing to easier movement of the

charge carriers and faster recovery of the chip. A fast

scan rate facilitated obtaining the gap at lower voltages

and acquiring the results in a shorter time.

To our knowledge, we demonstrate for the first

time the possibility to achieve and control the device

response in memristor measurement mode, using

external guidance: back gate for measurements in dry

conditions and microfabricated top gate for liquid

integrated experiments. The gating mimics the presence

of surrounding charged molecules, allowing the

opening or closing of the VGAP in a controlled manner to

set the initial conditions as needed. First, we performed

a dry conditions test using the back gate control, by

sweeping VSD between −2.5 and 2.5 V. Initially, no gap

opening was observed for zero or positive VG values

(Fig. 2(a)). Positive charges attract the negative carriers

at the channel region, facilitating their movement and,

therefore, the recovery of the conditions along the

sweep without any memory effect. Both current minima

crossed each other through the zero voltage value,

resulting in VGAP = 0 V. In contrast, a negative increase

of VG initiated the appearance of the characteristic

hysteresis in the I–VSD curve (Figs. 2(b) and 2(c)). The

separation between both peaks, i.e., the VGAP, reveals

an increasing tendency with VG increase. Once the gap

started to appear, VGAP dramatically increased with

small VG changes starting at approximately VG = −3 V,


5 Nano Res.

Figure 2 Voltage minima gap VGAP control with back gate in dry

conditions. (a)–(c) Drain-current–source-to-drain voltage I–VSD

curves at gate voltage VG from 0 to −3 V. As the VG decreases, the

VGAP widens. The current at the positive branch of the VSD sweep

also decreases, making the recovery more difficult, which results

in low currents at the negative branch of the backward sweep.

The orange region indicates VGAP. (d) Calibration of VGAP as a

function of VG for dry conditions. The gray area indicates the

most sensitive region, where the VGAP changes most dramatically

with the VG. The inset shows a cross-sectional diagram of the

device, where VG was applied via a back gate. (e)–(g) I–VSD

curves at various VG values in liquid conditions. (h) Calibration

of VGAP as a function of VG for liquid conditions (deionized

water). The gray area indicates the most sensitive region. The

inset shows a cross-sectional diagram of the device, where the VG was applied via the reference electrode in contact with the liquid.

as follows (see the grey region in Fig. 2(d))

GAP

G

4.5V

V

(2)

indicating a high sensitivity to changes in the surroun-

ding environment, as summarized in Fig. 2(d). Note

that the current at the positive branch of the sweep

decreased as the VG value became more negative,

owing to a decrease in the channel conductivity near

the source region. A large VG drop reduced more

electron carriers within the channel near the source

region. At the most negative VG value, the current was

low, and the device did not have enough time to

recover; this reflected as low current values even in

the negative branch of the sweep.

Next, the possibility to control the gap when a

liquid sample and the reference electrode were used

was tested by depositing a 100-μL deionized water

droplet. Liquid measurements confirmed the earlier

demonstrated trend in the gap opening as a function

of VG increase (Figs. 2(e)–2(h)), with the only difference

being that 10-fold lower VG were necessary, which was

attributed to the thin top oxide layer (5-nm thickness)

compared to the back oxide (200-nm thickness). By

defining the gate potential through the liquid

environment, the gap started to be seen at −175 mV,

with an abrupt hysteresis increase of

GAP

G

60V

V

(3)

in the most sensitive range (13-fold more sensitive

than when using a back gate, as indicated by the grey

region in Fig. 2(h)).

2.2 Bare memristor and transistor performance for

pH sensing

Considering that the application of a VG is analogous

to the presence of charged species of a certain sign,

the change in the charge content in a liquid solution

close to the nanowires’ surface should affect the VG

required to open the gap. Positive charges would

require the application of a more negative VG to com-

pensate their opposite effect, and vice versa. Phosphate

buffered saline (PBS) drops (100 μL), 100-fold diluted

and adjusted to various pH values, were deposited on

the sensors, and the VGAP was calculated at different VG.

As observed in Fig. 3(a), as the PBS solution became

more basic (i.e., less positive charges/protons), the VG

needed to be increased to more positive values to open

the gap. For acidic samples, the VG needs to be increased

by 50 mV/pH to maintain a VGAP value of 1.5 V


6 Nano Res.

(Fig. 3(b)), while the sensitivity reduces to 7 mV/pH

in the basic range.

A comparative test using the sensor in the traditional

FET mode (Figs. 3(c) and 3(d)) shows the expected

behavior, with a shift of the transfer curve to the

positive direction as the pH increases. By measuring

in the subthreshold regime (at I = 0.01 μA), a sensitivity

of 35 mV/pH was obtained in the most sensitive acidic

range, which slightly decreased, as expected, for the

basic range (7 mV/pH). The larger amount of mV/pH

needed to maintain VGAP = 1.5 V in the case of the

memristor mode could be a sign of its higher sensitivity

compared to that of the FET mode. However, the loss

of linearity for the smaller VGAP values and the crossing

of the different curves indicate that the control of this

mode requires further studies to improve it.

2.3 Specific Ebola detection using bio-memristor

Finally, we applied the memristor mode for the

specific detection of the Ebola VP40 matrix protein

in a liquid phase and compared it with that using

the FET operation mode. Specific antibodies against

the VP40 protein were immobilized onto the amino

modified nanowires’ surface, using carbodiimide

chemistry. The steps followed are shown in Fig. 4(a)

and explained in more detail in the Experimental

section. Briefly, the bare nanowires were plasma

activated to generate hydroxyl groups, onto which

3-aminopropyl triethoxysilane (APTES) was attached

by incubating for 1 h in an ethanolic solution. After

cleaning the surface with ethanol and drying the

devices for 30 min at 120 °C, the antibodies were

immobilized on the APTES-modified surface by

incubation for 1 h in 1× PBS in the presence of 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide hydrochloride

(EDC) and N-hydroxysuccinimide (NHS), followed

by a final rinse with PBS and a blocking step of

the remaining free surface sites with bovine serum

albumin (BSA) (0.5 mg/mL). More details on the

biofunctionalization can be found in the experimental

section.

For the analysis of the biosensing response, PBS

droplets (20 μL) with increasing VP40 concentrations

were incubated for 30 min on the biosensors, and then

cleaned with PBS to remove loosely bound proteins.

After rinsing with sodium phosphate buffer (SP) at a

5-mM concentration, a 100-μL droplet of the SP buffer

was deposited to obtain an increased Debye length

and thus higher sensitivity during the measurements.

The signals using both memristor and FET approaches

Figure 3 Calibration of the sensor sensitivity toward different pH solutions. (a) and (b) Using the memristor approach; (c) and (d)using the field-effect transistor approach.


7 Nano Res.

were acquired and compared.

Figures 4(b) and 4(c) show the response of the

biosensor following the memristor approach. A shift

of the VGAP–VG curve to the right could be observed with

increasing antigen concentration, indicating molecule

detection at femtomolar levels, with 6 fM being the

smallest detected concentration. The detection range

and the smallest detected concentration was the same

when using the FET measuring mode, although the

signal change was more pronounced in the latter case

(Figs. 4(d) and 4(e)), with a shift of the transfer curve

toward higher VG values. A specificity test was done

using staphylococcal enterotoxin B (SEB). Toxins pro-

duced by this bacterium cause similar initial symptoms

to those of Ebola (i.e., fever, diarrhea, vomiting) [50].

The results, depicted in Fig. 4(e), showed that the

deviation of the VG caused by nonspecific binding

was significantly lower.

These results agree with the detection capabilities

of the honeycomb patterned FETs, as published

previously [51], and improve the picomolar detection

level expected for rapid Ebola diagnosis in point-of-care

situations [52]. Furthermore, the detection range is

similar to that of another recent Ebola nanosensor,

where detection was possible at 50 fM, although

still requiring labeling for optical detection [46]. A

comparison of different Ebola detection techniques is

summarized in Table 1.

As a reference measurement and for comparison,

the VP40 assay was performed using the standard

ELISA format to confirm the immunorecognition of

the antigen by the antibody and to compare the results

of our devices with those of a traditional and well-

established technique that relies on optical transduction.

The antibody-antigen recognition was carried out using

horseradish peroxidase (HRP)-modified antibodies.

Figure 4 Sensor surface functionalization and results of the VP40 detection using such surfaces. (a) Functionalization steps: (i) bare honeycomb nanowires, (ii) silanization with APTES, (iii) antibody immobilization, and (iv) detection of the VP40 protein. (b) and (c)Calibration of the biosensor by VP40 detection in memristor mode. The inset in (c) shows the optical detection of VP40 by the enzyme-linked immunosorbent assay technique using horseradish peroxidase-modified antibodies. (d) and (e) Calibration of the biosensor by VP40detection in field-effect transistor mode, including the specificity test with the SEB (black data points).


8 Nano Res.

Incubation for 10 min in tetramethylbenzidine (TMB)

resulted in the development of a blue color that turned

to yellow upon addition of a stop solution (0.2 M

sulfuric acid). The absorbance was obtained at 450 nm

using an ELISA multiwell reader. The result is shown

in the inset of Fig. 4(e). Measurable signals could be

obtained from the antibody–antigen binding events,

with a linear increase of the absorbance as the amount

of antigen increased up to 50 nM, and slowly saturating

afterwards. The smallest concentration that could

be sensed accurately was 6.25 nM. The two electrical

readout modes, memristor and FET, outperformed

the traditional ELISA technique in both detection

capabilities (by six orders of magnitude) and

miniaturization.

3 Conclusions

A gate-controlled bio-memristor based on a large

nanowire area patterned in a honeycomb structure

and capable of sensing biomolecules directly in liquid

environments is presented here for the first time. The

gate control is used to engineer the opening of the VGAP

during the measurements, allowing initial conditions

that permit the detection of analytes of any charge

sign to be set. The biosensing capability of this device

at the femtomolar level is shown for the Ebola VP40

matrix protein, which is better than the results of

both the traditional method (ELISA) and other recent

nanosensors [46]. The biosensor can perform highly

sensitive, label-free measurements in two ways: as

an ion-sensitive FET and through a violation of its

memristor zero-crossing signature by opening a VGAP.

The memristor approach—a recent method—has

been demonstrated directly on liquid samples, which

would allow its application in a more simplified

setup that does not require humidity control. The

controllability of the VGAP using external guidance

through the gate electrode has been demonstrated on

a honeycomb nanowire pattern, rather than on a

single or a few linear nanowires [37, 38]. Furthermore,

the engineering of the VGAP opening (the basic property

used for the quantification) has been demonstrated in

dry (through the back gate) and liquid (through the

liquid gate, using the reference electrode) conditions.

The measurements can then be performed either by

measuring the VGAP change at a fixed VG or by measuring

the changes in the VG needed to maintain a fixed VGAP.

We predict that this device can be of great importance

for improving the control of memristor biosensors

in the ultrasensitive detection of both positively and

negatively charged molecules in realistic point-of-care

situations and for the rapid diagnosis of fast-spreading

diseases.

Table 1 Comparison of different Ebola detection techniques

Technique Target Lowest detection Miniaturization Particular drawbacks

Optical (plasmonic) [44] Live virus 106 pfu/mL + Special safety facilities

Surface acoustic wave [45] Whole virus 104 pfu/mL +

Fluorescence (flow cytometry) [53] Whole virus (DNA staining)

105 pfu/mL – – Fluorescent labeling required

Plaque assay [53] Live virus 101 pfu/mL – – Special safety facilities, several days duration

Quantitative reverse transcription-polymerase chain

reaction (qRT-PCR) [53]

RNA 103 pfu/mL – – Special training and equipment, hours duration

TEM [53] Virus particles (VP) 106 VP/mL – – Special equipment, hours duration

Optical (nanoparticle based luminescence) [46]

DNA fM – – Nanoparticle labeling required

ELISA (optical, absorbance) (inset Fig. 4(e))

VP40 nM – – Labeled antibody for colorimetric enzymatic reaction required

This work (memristor/FET) VP40 fM + +


9 Nano Res.

4 Experimental

4.1 Device fabrication

Devices were fabricated on silicon-on-insulator wafers

(p-type, ~10 Ω·cm) with 200-nm-thick oxide and

50-nm-thick top silicon layers. Phosphorus at a concen-

tration of 1014 cm−2 was implanted in the top layer.

The dopant was activated by rapid thermal annealing

at 1,000 °C. The active region was formed and isolated

using inductively coupled plasma reactive ion etching

(ICP-RIE). Then, a heavy implantation of phosphorus

was placed into the source and drain region, followed

by dopant activation by rapid thermal annealing

at 1,000 °C. The honeycomb pattern of 50-nm-wide

nanowires was defined by electron beam lithography

and ICP-RIE. The total sensing surface area of the

nanowires was 365.2 μm2. A gate oxide layer of 5 nm

was grown using a thermal furnace at 900 °C. A

transmission electron microscope (TEM) observation

revealed that the etching and oxide growth processes

resulted in final nanowire widths of 40 nm at the

bottom and 25 nm at the top (see bottom inset in

Fig. 1(g)). Subsequently, stacked 50-nm Ti and 200-nm

Ag layers were evaporated to form the reference

electrode as well as the external pads for source and

drain. The purpose of the reference electrode was to

define the potential of the liquid samples. It was used

as the top liquid gate for the measurements in liquid

conditions.

The devices were isolated by spin coating a 2-μm

SU-8 resist. The active region (including the nanowire

channel region and the reference electrode) were left

exposed to the samples. The structure of the reference

electrode was completed by an electrochemical reaction

in 0.1 M KCl. A droplet of the buffer was deposited

on the electrode, and 1 V bias was applied from the

electrode in the presence of a grounded platinum

wire. The reaction created an Ag/AgCl layer on top of

the Ag electrode.

As a result of the fabrication process, n-type HC-FETs

(Fig. 1(e)) were obtained, with a nanowire region that

was 10-μm long and 150 μm away from the reference

electrode, as described previously [49].

4.2 Silicon nanowire biofunctionalization

A widely used biofunctionalization procedure was

followed for the immobilization of the antibodies on

the nanowires’ surface. As shown in Fig. 4(a), first,

air-plasma activation was applied to increase the

hydroxyl group content, as anchoring points for the

molecules immobilized in the next step. Then, a

silanization process was carried out using APTES.

The plasma-activated devices were immersed for 1 h

in an ethanolic solution containing 2.5% APTES and

5% deionized water. After rinsing with ethanol, they

were dried in an oven at 120 °C. The antibodies were

then directly immobilized onto the reactive amino

groups of APTES by incubating them in 0.1 mg/mL

PBS for 1 h in the presence of 10 mM EDC and 5 mM

NHS to allow activation of the carboxylic groups in the

antibodies. The unbound antibodies were removed

by rinsing with PBS, and the remaining free surface

was blocked against nonspecific adsorption by

incubation in 0.5 mg/mL BSA in PBS. After a final rinse,

the devices were ready and kept in PBS at 4 °C until

their use for the biosensing experiment.

4.3 ELISA procedure for VP40 detection

First, antibodies were labeled with HRP, using the

Lightning-Link HRP conjugation kit (Innova Biosciences,

Cambridge, UK). This kit enables the modification of

antibodies by using a modifier for direct labeling

with HRP, with the further addition of a quencher to

stop the reaction and obtain the conjugated antibodies

without the need of any separations.

Then, the ELISA test was conducted. For this test,

different antigen concentrations were adsorbed in

a multiwall plate by incubation in a carbonate/

bicarbonate buffer, pH 9.3, for 1 h. After rinsing to

remove the loosely bound antigen molecules, the

unoccupied areas of the plate wells were blocked by

incubation in a PBS solution with 2% BSA and 0.05%

Tween 20 for 1 h. Then, the wells were rinsed again

and incubated with antigen-specific, HRP-labeled

antibodies in PBS. After a final rinse to remove unbound

antibodies, the TMB color substrate was added for

10 min. HRP oxidized the TMB, resulting in a blue

color, which changed to yellow after the addition of

0.2 M sulfuric acid as a stop solution. The absorbance

at 450 nm, which depended on the concentration of

bound antibodies, and, therefore, of adsorbed antigens,


10 Nano Res.

was measured using a Tecan microplate reader (Tecan

Group Ltd., Männedorf, Switzerland).

Acknowledgements

This work was financed via the German Research

Foundation (DFG) within the Cluster of Excellence

“Center for Advancing Electronics Dresden (CfAED)

EXC 1056” and the “ICT Consilience Creative Program”

(No. IITP-R0346-16-1007) supervised by the Institute

for Information and Communications Technology

Promotion (IITP), Republic of Korea.

Electronic Supplementary Material: Supplementary

material (optimization of the drain-to-source voltage

sweeping rate) is available in the online version of

this article at https://doi.org/10.1007/s12274-017-1720-2.

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