Integrating Microwave Resonator and Microchannel with

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Biological and Medical Applications of Materials and Interfaces

Integrating Microwave Resonator and Microchannel with Immunochromatographic Strip for Stable and Quantitative Biodetection Hong Zhou, Cheng Yang, Donglin Hu, Shaoxu Dou, Xindan Hui, Feng Zhang, Cong Chen, Ming Chen, Ya Yang, and Xiaojing Mu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02087 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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ACS Applied Materials & Interfaces

Integrating Microwave Resonator and Microchannel with Immunochromatographic Strip for Stable and Quantitative Biodetection Hong Zhou,1 Cheng Yang,3 Donglin Hu,1 Shaoxu Dou,1 Xindan Hui,1 Feng Zhang,1 Cong Chen,1 Ming Chen,3, * Ya Yang,2, * Xiaojing Mu1, * 1

International R & D center of Micro-nano Systems and New Materials Technology, Key

Laboratory of Optoelectronic Technology & Systems, Ministry of Education, Chongqing University, Chongqing 400044, China 2

Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, Beijing

100083, China 3

Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University

(Army Medical University), Chongqing 400038, China

KEYWORDS: Flexible material; Microwave sensing; Biomolecular detection; Staphylococcus aureus; Lateral flow immunochromatographic strip.

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ABSTRACT Immunochromatographic strip is an effective diagnostic tool in various fields due to its simplicity, rapidity and cost-effectiveness. However, typical strips for preliminary screening provide only qualitative or semi-quantitative results, and common solutions for quantitative detection by incorporating kinds of nanoparticles as biomarkers still do not solve this problem thoroughly. Here we try to tackle this challenge by integrating low-cost membrane compatible square split-ring resonators and structure-design-flexible microchannels with flexible strips. We experimentally demonstrate that the limit of detection (LOD) and sensitivity of the strip for quantitative detection of Staphylococcus aureus reach 0.784 ng/mL and 10.214 MHz/(ng/mL), respectively. The LOD level is about 63 times higher than that of the color-based strip determined by naked eye, and the stability is about 18 times higher than that of the fluorescent strip. This work could not only provide a powerful diagnosis tool for the quantitative detection of S. aureus or other molecules, but also deliver new avenues for achieving electric field detection of biomolecules, system-level integration of biosensors, and the development of portable diagnostic devices.


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1. INTRODUCTION In recent years, there has been an increasing demand for the detection of various biomolecules such as biomarkers,1 proteins,2 and drugs3 in biomedicine, as well as chemical contaminants in food safety and environmental monitoring.4-5 To date, numerous studies regarding the detection of biomolecules have been reported, including surface plasmon resonance (SPR),6-7 electrochemiluminescence (ECL),8 high performance liquid chromatography (HPLC),9

enzyme-linked

immunosorbent

assay

(ELISA),10

and

lateral

flow

immunochromatographic strip (LFICS).11-13 Among them, LFICS is widely used due to its simplicity, rapidity (detection within 10 minutes) and low cost. However, early LFICS used for preliminary screening provided only qualitative ("positive/negative" signals) or semi-quantitative results without quantitative information.14 In addition, low signal intensity and poor sensitivity also hinder the development of this simple detection method. Therefore, it is essential to improve LFICS to realize rapid, simple, low-cost and high-sensitivity quantitative detection of biomolecules. In general, quantitative detection with high analytical performance based on LFICS is mainly achieved by means of optical markers, including colored (e.g., colloidal gold, carbon and colloidal selenium nanoparticles),15-16 and luminescent (e.g., quantum dots, up-conversion phosphor nanoparticles and dye-doped nanoparticles) markers.17-19 For example, Xu et al. developed a gold-nanoparticle-decorated silica nanorod as a colored label for protein detection with a LOD of 0.01 ng/mL, which greatly improved the sensitivity of the traditional LFICS.20 Li et al. presented a quantum dot nanobeads-based sandwich LFICS for prostate specific antigen (PSA) detection, which resulted in a 12-fold enhancement in the LOD compared with that of conventional quantum dot strip.21 These studies demonstrate the effectiveness of optical markers in improving the sensitivity of LFICS. However, these methods require a strip/fluorescence reader and can only obtain information on the surface of the test line (about 10 μm thickness), 3 ACS Paragon Plus Environment


resulting in 90% of the analyte in the line being undetected. In addition, there is much interference in optical measurements, including scattering, self-absorption and specular reflection, as well as long-term signal quenching.22 As such, a new method that eliminates optical interference and provides stable and comprehensive detection of all targets in test line is required. Metamaterials was introduced by Victor Veselago in 1968,23 which are rationally designed artificial structures for controlling and manipulating electromagnetic and acoustic waves, leading to various exciting electromagnetic functionalities and applications. Recently, the development of metamaterial based sensing technologies has attracted great attention for the detections of biological and chemical analytes because of its high sensitivity, rapidity, cost effectiveness and comprehensive detection.24-29 Lee et al. fabricated a metamaterial based biosensor with split-ring resonators and used it to detect the binding of biotin and streptavidin.30 Subsequently, they developed a local high-impedance microstrip system to improve sensitivity, which enable the detection of PSA biomolecules at lower concentrations (LOD = 100 pg/ml) compared with that of previous studies.31 Kim et al. proposed a biosensor with a rectangular meandered line (RML) resonator on a gallium arsenide substrate by integrated passive device (IPD) technology, achieving the quantitative detection of the glucose in human serum.32 Although these studies demonstrated that the metamaterial technology is feasible for detecting biomolecule binding without the disadvantages of optical measurements mentioned above, the substrates of these devices are not flexible materials, which means the failure of the combination between metamaterial technology and LFICS. Fortunately, with the development of fabricating technology, low-cost paper-based metamaterial sensors have emerged and provide potential opportunities to integrate them. Tao et al. fabricated a terahertz subwavelength structure on the flexible paper using a shadow mask technique for quantitative analysis in biochemical sensing applications.33 More recently, Sadeqi et al. combined the microfluidic channel and microstructure on the flexible paper employing a wax patterning technique, realizing the 4 ACS Paragon Plus Environment

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detection of chemical molecules in the terahertz spectrum.34 However, these approaches can only yield a limited area of microfluidic channels for molecular detection, not exceeding square micron level, and are incompatible with the fabrication process of LFICS. Therefore, highly sensitive, low-cost, and process-compatible metamaterial-based biosensors integrated with LFICS for quantitative detection of biomolecules still present a challenge. Herein, we report the first LFICS integrated with planar square split-ring (SSR) resonator and microchannel, which overcomes the challenge associated with process incompatibility through the photoresist-free shadow mask deposition and wax printing techniques. Then, the metamaterial-based LFICS (meta-LFICS) is used to quantitatively detect the nucleic acid of staphylococcus aureus (S. aureus), which account for the majority of episodes of bacteremia in critically ill patients in the intensive care unit.35 The implementation of analyte detection depends on the ingenious cooperation of the two parts of this meta-LFICS, i.e., the lateral flow immunochromatographic component for maintaining immunochromatography in the membrane, and planar SSR resonator fabricated on the membrane for sensing the binding of biomolecules during immunochromatography. Since the biomolecules in the stripe are directly probed by the electric field excited by the resonator, this method eliminates the disadvantages of interference existed in conventional optical measurement, such as scattering, self-absorption and specular reflection, and can comprehensively detect all molecules in the test line. Importantly, an added advantage of high sensitivity and stability is experimentally demonstrated. This work will primarily focus on the detailed investigation of the meta-LFICS from the perspective of electromagnetic physics and biological detection. We believe that these findings will provide both a powerful diagnosis tool for the quantitative detection of S. aureus and a solution for successful system-level integration of biosensors.

2. MATERIALS AND METHODS 2.1. Design of the meta-LFICS 5 ACS Paragon Plus Environment


The proposed meta-LFICS, illustrated in Figure 1, is composed of three structural components: a lateral flow immunochromatographic component, a radio-frequency (RF) SSR resonator, and backing plate. The lateral flow immunochromatographic component consisted of sample pad, conjugate pad, test line (T-line), control line (C-line) and absorbent pad (See the inset A’-A’ of Figure 1a). In order to control the flow of the analyte towards the sensing area, wax printing techniques are employed to create hydrophobic barriers and hydrophilic channels. The SSR resonator is then designed on the membrane as a sensitive element for sensing biomolecules in the channel. The backing plate made of Polyvinyl chloride (PVC) functions as a support material for the system to prevent distortion and degradation of the membrane.

Figure 1. Schematic illustration of (a) the meta-LFICS integrated with the SSR resonator and the microchannel. Inset: the lateral flow component for maintaining immunochromatography, including sample pad, conjugate pad, test line, control line and absorbent pad. (b) Electric field and surface current distribution of SSR resonator obtained by using the finite element simulation software ANSYS HFSS. (c) The sensitive part of the SSR resonator with split capacitance (Cs). (d) The equivalent circuit of the SSR resonator involving Rs, Ls, Cs and ∆C, after the target analyte was attached to the split of the SSR resonator. (e) The corresponding shift of the transmission spectra, when there is a change (∆C) in split capacitance (Cs).

The detection of target analyte involves two steps. First, the immunochromatographic testing 6 ACS Paragon Plus Environment

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was performed on the lateral flow immunochromatographic component as in the conventional LFICS. When the sample was loaded onto the sample pad, it moved forward along the membrane through sample pad, conjugate pad, test line, and control line under the action of the capillary force. Once the sample flowed onto the T-line, the target molecule was captured by the antibody immobilized on the T-line due to specific binding, which would change the dielectric property of the membrane and induce a dielectric perturbation to the nearby SSR resonator. Then, the SSR resonator integrated on the strip sensed the dielectric perturbation by exciting an electric field. Figure 1b depicts the electric field and surface current distribution of the SSR resonator when electromagnetic waves are incident onto the meta-LFICS. Obviously, the energy of the electric field is mainly concentrated at the split of the resonator, up to 3×105 V/m, meaning that the split of the resonator must be aligned with the T-line to detect biomolecules more sensitively. In this way, all target molecules in the T-line can be detected by the strong electric field, thereby achieving strong signal and high sensitivity. Additionally, since the incident magnetic field is polarized perpendicular to the plane of the resonator, according to Faraday's law, the current in the resonator is a clockwise circulating current, as plotted in Figure 1b. The energy generated by the circulating current is stored across the gap, where the split of the resonators acts as a capacitor. Therefore, the capacitance of the resonator is described by: eff Cs   sub A d

(1)

eff

where  sub is the effective permittivity of the substrate, and A represents the area of the resonator.36 When the target molecules are captured by the antibody on the T-line, there will be a change in the capacitance of the resonator, denoted as ∆C, as shown in Figure 1c. Similarly, it can be expressed as eff A d C   sam

(2)

eff where  sam is the effective permittivity of the target biomolecules. It is clear from equation (2)



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eff that ∆C is determined by  sam because it is hard to change A and d after fabrication. In order to

model the entire working process of the system, an equivalent circuit is established, as shown in Figure 1d, which consists of a parallel LCR circuit. Thus, the resonant frequency of the system without target biomolecules is predicted as

f0  1 2 LsCs

(3)

where Ls represents the inductance of the resonator. The resonant frequency of the system with target biomolecules is written as

f '  1 2 Ls  Cs  C 

(4)

Then, the frequency shifts Δf =f′ -f0. As Ls is determined by the metal loop and remains unchanged after fabrication, the resonant frequency Δf is determined by ∆C. To demonstrate this theory, the equivalent circuit is simulated by the circuit analysis software Advanced Design System (ADS). The simulated results show that the resonant frequency of the resonator f0 = 7 GHz (Figure 1e). When the target molecules bind onto the T-line and cause ΔC = 30 pF, the resonant frequency f0 drifts accordingly and the frequency shifts Δf = 300 MHz, which is consistent with the above theory. Therefore, it is theoretically reasonable to choose Δf as an indicator for quantitative detection of S. aureus nucleic acid. 2.2. The fabrication of hydrophilic channels and hydrophobic barriers In order to create a hydrophilic channel for immunochromatography and control the flow of analyte to the sensing zone, wax-printed techniques were proposed to fabricate the hydrophilic channel and hydrophobic barriers. Figure 2a and 2b illustrate the process of fabricating channels and barriers on the membrane. In the absence of wax, the aqueous solution can easily flow along the fibers of the nitrocellulose membrane (NC membrane) due to the microstructure of the intertwined fibers (Figure 2a1 and 2a2). When the wax is printed and melted onto the membrane, the wax-covered fibers were converted into hydrophobic barriers, resulting in the formation of 8 ACS Paragon Plus Environment

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microchannel structures (Figure 2b1 and 2b2). The detailed fabrication process of microfluidic channels mainly involves two steps (See Section S6 in Supporting Information): (1) printing the channel microstructure onto the surface of the NC membrane using a wax printer; (2) the wax-printed membrane was baked in an oven at 120°C for 5.5 minutes. The purpose is to melt the surface wax and penetrate through the membrane to form hydrophobic barriers. The entire process can be completed in 10 minutes. It is worth noting that the baking temperature should be carefully controlled accordingly to protect the highly flammable membrane (flash point 200 °C). 2.3. The fabrication of the SSR resonator As the sensing element of the system, the SSR resonator was integrated onto the patterned membrane by magnetron sputtering, and its fabrication process is shown in Figure 2b and 2c. The detailed fabrication process of the resonator mainly includes two steps (See Section S7 in Supporting Information): (1) A 0.1 mm thick SUS304 plate was cut by a laser and then electrolytically polished to form a shadow mask of high precision and smooth surface with a minimum spacing of 0.01 mm. (2) The SSR resonator was formed by sputtering a 200 nm thick Au onto the surface of the membrane through the above shadow mask. It is worth noting that a cross mark needs to be designed in the shadow mask to align the resonator with the microfluidic channel. Additionally, Au is deposited on the surface of interwoven fibers because the dimension of the Au particles is smaller than that of the pores of the membrane (Figure 2c1). Since the three-dimensional interwoven fibers structure of the membrane remains unchanged and the Au particles penetrate only the surface of the membrane, the channel remains hydrophilic and suitable for liquid flow, as shown in Figure 2c2.



Figure 2. Fabrication procedures of the meta-LFICS. (a)-(b) Hydrophilic channel and hydrophobic barrier were formed by wax-printed technology. (c) The SSR resonator was integrated onto the printed membrane by magnetron sputtering. (d) T-line and C-line were drawn in hydrophilic channel by a dispenser. (e) The biomolecule adsorbed membrane was cut into sensor elements for the subsequent preparation of LFICS. (f)-(g) Sample pad, conjugate pad, absorbent pad and backing plate were assembled together into the meta-LFICS. (h) Photograph of the meta-LFICS. Upper panel: the dimension of the resonator and membrane. (a1)-(c1) Cross-section optical images of strip in step (a)-(c), Insets: the corresponding enlarged images. (a2)-(c2) Schematic diagram of corresponding cross-section when filled with aqueous solution.

2.4. The preparation of the lateral flow component Figure 2d-2g show a schematic illustration for the preparation of the lateral flow immunochromatographic component. Remarkably, three processes including printing, sputtering and spraying are carried out on the same membrane before cutting. In this way, dozens of strip elements can be prepared from one membrane (Figure 2e), which is beneficial to maintain consistency in each strip. The detailed preparation process of all components is as follows: Sample pad was prepared by using PBS solution (0.015M, pH 7.2, containing 0.1 mol/L NaCl, 10 ACS Paragon Plus Environment

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0.2% (v/v) Tween-20, 2% (w/v) Bovine serum albumin (BSA)), and then dried in a drying oven of 37 °C for 30 minutes. The Au-streptavidin was added to the PBS solution (0.015M, pH 7.2, including 0.6mol/L NaCl, 3% (w/v) sucrose, 0.2% (v/v) Tween-20, 2% (w/v) BSA), and then loaded onto the conjugate pad through a dispensation system. After that, it was dried in a drying oven of 37 °C for 30 min and stored at 4 °C until later use. Afterwards, according to optimization, 50 times anti-fluorescein isothiocyanate (FITC) antibody and 50 times biotin labeled BSA (BSA-Biotin) were dispensed onto two separate areas of the hydrophilic channel to prepare T-line and C-line (Figure 2d). The T-line must be aligned with the split of the resonator to improve detection sensitivity. The absorbent pad was employed to absorb liquids and maintain the capillary force in the membrane, and the PVC plate served as a support material. All components including the sample pad, the conjugate pad, the NC membrane and the absorbent pad were sequentially assembled to the PVC plate, and each component overlapped 1.5 mm at the junction, as shown in Figure 2f and 2g. After assembly, the strips were stored in a plastic box until later use. Figure 2h exhibits the optical photograph of the meta-LFICS after fabrication, whose overall size was 1.2 cm × 6 cm.

3. RESULTS AND DISCUSSION 3.1. Characterization of hydrophilic channels and SSR resonators Wax-printed technology has several attractive features such as high-efficiency, low-cost, simplicity and high-flexibility, providing a promising approach to the fabrication of microfluidic devices. Hydrophilic channels prepared by this wax-printing technique can be characterized in three ways: the microstructure of the membrane, the hydrophobicity of the barriers, and the precision of the channel width. To characterize the microstructure of the membrane, it was observed by scanning electron microscopy (SEM). Generally, the microstructure of the NC membrane was networks of interwoven cellulose fibers with many pores suitable for lateral flow of liquid (Figure 3a and 3d). The surface roughness of the hydrophilic paper substrate (Figure 3g) 11 ACS Paragon Plus Environment


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was characterized atomic force microscopy (AFM). When the membrane was covered by wax, the three-dimensional (3D) structure of the NC membrane maintained an interwoven network structure during wax printing and baking (Figure 3b and 3e). However, the membrane turned to be hydrophobic due to the hydrophobic wax. Its hydrophobic properties were characterized by the contact angle (CA), which were measured by contact angle analyzers. The CA on the front and back of the membrane were 126° and 109°, respectively, as shown in Figure 3h. The CA of the front side was larger because the wax melted and penetrated from the front side to the back side. As for the channel width, since the wax penetrated both vertically and laterally during the baking process, the width of the channel was different before and after baking. The flow of wax is a capillary flow, which can be estimated by the Washburn equation:37

l

 rt cos  4

(5)

where η ， γ, θ and l represent the viscosity of the liquid, the surface tension, the CA and the distance penetrated through a porous membrane with an average pore radius r, respectively. It can be inferred from equation (5) that the spreading distance l is proportional to time t, after the device is fabricated to keep other parameters constant. The final width of the channel is equal to the printed width minus the distances caused by the flow of wax. To demonstrate this theory, channels with widths varying from 700 μm to 1400 μm were designed (100 μm increment per step). After wax was melted at 120 °C for 5.5 minutes, final channel widths were observed and measured with an optical microscope and its accompanying image software (See Section S5 in Supporting Information). The results revealed that the channel widths after baking were linearly related to the original channel widths, and then the spreading distance of front side was measured to be 162 μm. Therefore, considering the flow distance of the wax, the printing width of the channel was set to 1.162 mm. After baking, the final channel width was 1.01 mm (See Figure S1 in Supporting Information), which met the resonator's requirements for alignment.


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a

b

c

20μm

20μm

e

d

f

2μm

2μm

200 nm

g

h

CA=124°

i

Front

0.4 nm

Resonator

0 nm 10 um

20μm

8

6

4

2

2

4

6

8

10 um

Split

Resonator

CA=109°

Back

500μm

Figure 3. Characterization of the hydrophilic channel and the SSR resonator. SEM images of the (a, d) bare membrane, (b, e) wax covered membrane, and (c, f) Au covered membrane. (g) AFM micrograph showing details

of the hydrophilic channel. (h) The CA of wax covered membrane. (i) The optical micrograph of the split of the SSR resonator.

In order to obtain a high quality factor (Q factor) of the resonance, it is necessary to investigate the conductivity of the resonator on a microscopic scale. When a highly conductive Au was sputtered onto the surface of the membrane, the interwoven fibers were covered by Au particles, as exhibited in Figure 3c and 3f. Obviously, the Au particles were attached to the fibers and joined together to form a stable electrically conductive structure. The corresponding electrical resistivity of the Au layer was only 5.9 Ωm at 20 °C, indicating the effectiveness of the stable electrically conductive structure. In addition, since the outline of the split of the resonator was neat and regular, it is suitable for the storage of electrical energy, as shown in Figure 3i. The 13 ACS Paragon Plus Environment


final split width was 0.99 mm, which met the channel’s requirements for alignment. 3.2. Proof-of-concept demonstration of biomolecules sensing For the proof-of-concept demonstration of our meta-LFICS as biosensors, empty strips without any immobilization of biomolecules were prepared. The empty strips canceled the T-line and C-line, and the conjugate pads also did not immobilize any antibodies or biomarkers, as described in the inset of Figure 4a. Under these conditions, their resonant frequency is constant and only relevant to the design and fabrication processing. Figure 4a revealed the model, simulated and measured transmission spectra of an empty strip. Although the resonant frequency of the simulated result was consistent with that of the measured result, there was a difference between their amplitudes, which was due to fabrication errors and systematic errors in the measurement system. After fitting the calculated results of the equivalent circuit model to the measured results by the circuit analysis software Advanced Design System (ADS), the total electrical components of the resonator are obtained, i.e., Ls=269.18 pF, Rs=231.30 Ωm , Cs=1.90 pC. Figure 4b shows the resonant frequency of nine strips (denoted as 1# to 9#). The resonant frequencies of these strips were approximately 7.0 GHz, and the difference between the maximum (5#) and minimum (9#) frequencies was 60 MHz, which is only 0.86% of the average frequency. These differences are acceptable and can be improved by more sophisticated and expensive fabrication process. Glucose molecules were chosen as an analyte to investigate the sensing performance of these strips. The glucose solutions were prepared by dissolving glucose crystals in deionized water, and their concentrations vary from 0.5 mmol/L (9 mg/dL, severe hypoglycemia) to 30 mmol/L (540 mg/dL, severe hyperglycemia). Then, 50 μL of glucose solutions with different concentrations were loaded onto the channels of strips 2# to 9#. After that, the strips were allowed to stand for three minutes, air-dried in an incubator at 40°C for three minutes, and then measured in the experimental setup. The correspondingly measured transmission spectra of strips 14 ACS Paragon Plus Environment

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after loading glucose solutions are shown in Figure 4c. For higher glucose concentrations, the resonant frequency shifted more towards the low frequency direction because the shift was mainly induced by the change in the split capacitance and could be estimated by equation (2). As a validation, a maximum shift of 405 MHz was obtained by loading the 30 mmol/L glucose solution onto the strip 9#, while only a 10 MHz shift was observed for the 0.5 mmol/L glucose solution (Strip 2#). When pure deionized water was added onto the strip 1#, the correspondingly transmission spectra were acquired and compared before and after the addition (See the response of Strip 1# in Figure 4c). Obviously, there was no shift between them, which meant that the observed shifts on the strips 2# to 9# were attributed to the glucose molecules. When the reference frequency of each strip is set to the resonant frequency before loading the glucose solution, the frequency shift of glucose with various concentrations can be plotted in Figure 4d. It was clear that the frequency shift varied linearly with the concentration of glucose solution, and the higher the concentrations of the glucose solution, the more severe the frequency of strips shifted. The relationship between them is close to y = 13.2664 x + 8.3763 with a fitting degree (R2) of 0.9979. When the sensitivity is defined by the slope angle of the fitting curve, it reaches 1.32664×107 Hz/(mmol/L). The implementation of this glucose detection can be understood as follows (See the inset of Figure 4d): When the glucose molecules are attached to the cellulose of the membrane, the alterations in the dielectric constant of the membrane induce a change in the split capacitance, thus resulting in a frequency shift. According to Equation 4 and the experimental results on Strip 1#, the frequency shift is determined by the target glucose molecules. Therefore, it is reasonable to quantitatively detect biomolecules by utilizing the linear relationship between frequency shifts and biomolecule concentrations.



b

0 -5

S21(dB)

-10 -15

Empty Strip

Model Result Simulated Result Measured Result

-20 -25 6.0

c

6.4

6.8

7.2

d

-10 -12

Frequency Shift (MHz)

None 1# DI water 1# 0.5 mmol/L 2# 1 mmol/L 3# 3 mmol/L 4# 5 mmol/L 5# 7 mmol/L 6# 9 mmol/L 7# 20 mmol/L 8# 30 mmol/L 9#

-8

5.5

6.0

6.5

Freqency (GHz)

7.2

7.0

fmax= 60 MHz

7.0

6.8 1#

-2

-6

Resonant Frequency

7.6

Freqency (GHz)

0

-4

7.4

Resonant Frequency (GHz)

a

S21(dB)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2#

420

3#

4#

5#

Strip

6#

7#

8#

9#

Frequency Shift Fitting Curve

360 300

y = 13.2664x + 8.3763

240

R2 = 0.9979

180

ΔԐ

ΔC

Flow

120

Δf

C6H12O6

Glucose

60 0 0

7.5

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3

6

9

12 15 18 21 24 27 30

Concentration (mmol/L)

Figure 4. The proof-of-concept demonstration of biomolecules sensing using bare meta-LFICS. (a) The model, simulated, and measured transmission spectra of the empty strip. (b) Resonant frequency of strips 1# to 9#. Inset: Top view of the strip without any immobilization of biomolecules. (c) The measured transmission spectra of strips 1# to 9# when various concentrations of glucose are loaded into the channel. (d) The corresponding resonant frequency shift for glucose with a fitting curve of y = 13.2664 x + 8.3763. Inset: the schematic diagram of detecting glucose molecules using the proposed strip without any immobilization of biomolecules.

3.3. Mechanism of the meta-LFICS for quantitative detection of Staphylococcus aureus Although the strips have proven to be useful for the detection of biomolecules, there are still some challenges in the specific detection of target biomolecules. In our system, the S. aureus 16 ACS Paragon Plus Environment

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nucleic acid was captured by specific binding between the antigen and the antibody, and then it was quantitatively detected by electric field excited by the SSR resonator. The detailed specificity of the meta-LFICS is analyzed in Section S9 in Supporting Information. Figure 5 reveals the detection mechanism of S. aureus using the proposed meta-LFICS. First, 50 μL of positive samples (containing the nucleic acid of S. aureus modified by biotin and fluorescence) were loaded onto the sample pad (Figure 5aⅠ). The type of S. aureus is mentioned in Section S1 of the Supporting Information. S. aureus nucleic acid was modified by fluorophores for two reasons: 1) The S. aureus nucleic acid was captured on the T-line by specific binding between the anti-fluorescein isothiocyanate (anti-FITC) and the FITC-labeled nucleic acid. 2) The intensity of the fluorescence was detected by a fluorescence microscope to determine the amount of S. aureus nucleic acid, which could be used to compare fluorescence detection methods with our proposed methods. In addition, the S. aureus nucleic acid was modified to achieve colloidal gold labeling, which could be utilized for comparison between colloidal gold detection methods and our proposed methods. After loading, the target molecules in the samples flowed towards the conjugate pad, where the Au-streptavidin specifically bound to BSA-biotin and continued to flow toward the T-line (Figure 5aⅡ). Then, the target molecules were captured on the T-line by the specific binding between the anti-FITC and the fluorophore (Figure 5aⅢ). Finally, streptavidin without target molecule specifically binds to BSA-biotin on the C-line (Figure 5aⅣ), causing the C-line to turn red, which can determine the effectiveness of the test. When the target molecules are captured on the T-line, the split capacitance changes accordingly and causes shifts in the resonant frequency, which serves as an indicator of quantitative detection of S. aureus (Figure 5c).



Figure 5. Detection mechanism of S. aureus using the proposed meta-LFICS. Biological reaction in the lateral flow component when (a) positive and (b) negative sample were added to the meta-LFICS. Schematic diagram of molecular detection by the SSR resonator when the nucleic acid of S. aureus is (c) present or (d) absent on the test line. Inset: Corresponding frequency shift. Photograph of the meta-LFICS after (e) positive and (f) negative sample were loaded.

In the absence of target molecules (negative sample), the Au-streptavidin on the conjugate pad was carried by the liquid to the T-line (Figure 5bⅠand 5bⅡ). No molecules were trapped on the T-line due to the absence of target molecules in the liquid (Figure 5b Ⅲ ). Finally, Au-streptavidin specifically bound to BSA-biotin on the C-line (Figure 5b Ⅳ ). There are two differences between the tests for positive and negative samples: 1) The strip loaded with positive samples exhibits frequency shifts due to the presence of target molecules in the split of the 18 ACS Paragon Plus Environment

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resonator (Figure 5c), while the resonant frequency of the strip loaded with negative samples remains constant (Figure 5d), which are applied to quantify S. aureus. 2) For positive samples, red bands appear on both the T-line and C-line. However, for the negative sample, only the C-line emerges a red band. These attributes form the principle for quantitative detection of S. aureus. The detecting mechanism of the meta-LFICS is essentially different from that of the fluorescence-based strip. Conventional fluorescent strips achieve quantitative detection of target analytes by measuring the fluorescence intensity of markers using a fluorescence microscope. The differences mainly include: 1) fluorescence-based method achieves quantitative detection by means of optical markers, including colored and luminescent markers. In contrast, the meta-LFICS is a label-free method of detecting target analytes by electric fields; 2) fluorescence-based method requires a fluorescence microscope as a reader, while the reader for meta-LFICS is a radio-frequency device. In general, electronic readers have advantages in volume and cost compared to fluorescent microscopes; 3) conventional fluorescence-based method (excluding up-conversion luminescent strips) may suffer from interferences of scattering, self-absorption and specular reflection, which can be eliminated in the meta-LFICS. 3.4. Analytical performance of the meta-LFICS According to the above-mentioned mechanism, the meta-LFICS was applied to quantitatively detect S. aureus nucleic acid. Firstly, a series of S. aureus nucleic acid solutions with various concentrations (0.032, 0.16, 0.8, 1, 4, 20, and 100 ng/mL) were prepared by the method described in Section S3 of the Supporting Information. After preparation, the nucleic acids were amplified according to the method in Section S4 of the Supporting Information. The amplification process can be observed using the ViiA 7 real-time PCR system, as shown in Figure 6a. An equal volume of phenol:chloroform (1:1) was then added to the analytes at the 30th cycle and allowed to stand for 2 minutes. After addition, it was loaded onto our meta-LFICSs to investigate their analytical performance. The strips were allowed to stand for 19 ACS Paragon Plus Environment


three minutes, air-dried in an incubator at 40°C for three minutes, and then measured in the experimental setup. The experimental setup for testing is illustrated in Section S8 in Supporting Information, which is simpler and smaller than the optical instrument, especially when using a handheld device, the meta-LFICS can be a portable diagnosis tool for fatal diseases in undeveloped areas.

a

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Figure 6. The measured results of S. aureus using the proposed meta-LFICS. (a) The measured fluorescence intensity during recombinase aided amplification of nucleic acid. (b) The measured frequency shifts of the strips when the concentrations of S. aureus vary from 0 ng/mL to 100 ng/mL. The error bar plots are extracted from 4 different samples for each concentration, and the insets are the photo of T-lines for strips at various concentrations. (c) Fluorescence images of the T-line and C-line at various concentrations. (d) Comparison of the stability between the fluorescence intensity from the fluorescent strip and the frequency shift from our meta-LFICS.

For the detection of S. aureus solutions with various concentrations, the measured frequency 20 ACS Paragon Plus Environment

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shifts of the strips were shown in Figure 6b. The frequency shifts for the S. aureus solutions at concentrations of 0.032 ng/mL and 0.16 ng/mL were close to zero, and that for the 0.8 ng/mL sample was 5.1 MHz. Starting from this concentration, the frequency shifts are approximated as a linear response which ascends as the concentration of sample increases. This linear relationship can be described as y = 10.214 x + 25.95 with a fitting degree of 0.996. Therefore, the LOD of our device was calculated by the equation 3σ/S = 0.784 ng/mL, where σ and S represent standard deviation and slope of this linear relationship, respectively, and the sensitivity for the detection of S. aureus nucleic acid reached 10.214 MHz/(ng/mL). It was worth noting that the LOD of the real-time polymerase chain reaction (RT-PCR) was better than the meta-LFICS. The implementation of high LOD relies on rigorous experimental conditions, including complex temperature cycle, vibration-free environments, and expensive reagents and instruments.38-39 Therefore, the advantages of RT-PCR include high sensitivity and commercialized technology, and this method is mainly used in large hospitals and laboratories because of the demand for expensive reagents and instruments. The advantages of this work compared with RT-PCR include: 1) the cost of the reagents for meta-LFICS is lower than that for RT-PCR; 2) the reader for the meta-LFICS is simpler and cheaper than that for RT-PCR; 3) as a LFICS, the operation of the meta-LFICS is much simpler and the detection time is shorter. It takes a total of 5 minutes from the start of loading the target molecule to the end of the test result. In addition to the analytical performance, we also investigated the superiority of the meta-LFICS compared to two kinds of traditional LFICS, i.e., color-based strips and fluorescent strips. Color-based strips depend on the color change of colloidal gold on the T-line to determine the level of sample concentration, and it is widely used because of its simplicity, rapidity and visual interpretation. However, naked eye determination concluded that the LOD was 50 ng/mL, which exposed its fatal shortcoming of the poor sensitivity, as exhibited in the inset of Figure 6b. For the fluorescence-based method, the concentration of the sample is mainly determined by the intensity of the fluorescence, but it is not easy to distinguish the low fluorescence level with the 21 ACS Paragon Plus Environment


naked eye, indicating the necessity of the fluorescence reader in this method. Figure 6c presents the measured results of samples with various concentrations by a fluorescence reader equipped with analysis software. Starting from the concentration of 0.1 ng/mL, the fluorescence intensity increased with the increment of nucleic acid concentration, so 0.1 ng/mL was determined as the LOD of this method. Therefore, the fluorescent strip has the same level of LOD as our meta-LFICS. However, there are some unavoidable disadvantages in the fluorescent strip, such as toxic, fluorescence quenching, and bad chemical and colloidal instability [26], which will be analyzed in the later section. The comparative analysis of the performance between the meta-LFICS and previously reported biosensors is summarized in Table S2 in Supporting Information. 3.5 Stability analysis of the meta-LFICS As a critical indicator in evaluating sensor performance, the stability of the strip determines the scope of its application and the reliability of its detection results. Therefore, we further investigate the stability of the meta-LFICS from two aspects, i.e., measurement error and fluctuation of detection results over time. There are two main types of measurement error sources in the strip, including random errors and systematic errors. As the random errors are caused by factors that randomly affect measurement of the variable across the sample, it is usually reduced by analyzing more observations and averaging them. In this study, four different samples at each concentration were measured and the points derived from the measurement were plotted in the Figure 6b to extract standard deviation as error bars. The bars reveal the range of random errors during the test without affecting the approximate linear relationship between concentration and frequency shift. In addition, the standard deviation reflected the matrix effect in the meta-LFICS (See Section S11 in Supporting Information for more details). The systematic errors in experimental results usually come from the measuring apparatuses, so the test system must be calibrated by Calibration Kit (Keysight Technologies, USA) before the experiment to 22 ACS Paragon Plus Environment

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reduce it. In order to investigate the changes in test results with respect to time, the positive samples are simultaneously loaded onto the fluorescent strips and our meta-LFICS, and then the results are obtained every 10 minutes. Figure 6d exhibits that the fluorescence intensity drops sharply first and then gradually decreases to a low level, but the frequency shift of the meta-LFICS descends little and remains constant around 95 MHz. When the rate of change ∆r is defined as ∆α/β, where β and ∆α represent the initial and variation values of fluorescence intensity or frequency shift, the ∆r of LFICS (5.0%) is 18 times lower than that of the fluorescent strip (90.9%), which means that our meta-LFICS possesses a more stable detection capability compared to the conventional fluorescent strip. This interesting phenomenon can be explained in two ways: 1) continuous illumination of ambient light on the FITC may cause oxidation of the surface and photobleaching, resulting in fluorescence quenching; 2) the label-free detection of biomolecules by the strip is determined by the intrinsic dielectric properties of the analyte, which eliminates some of the instability factors such as quenching and decomposition. In addition, it is roughly estimated that the proposed strip has a shelf life of 12 months (See Section S10 in Supporting Information for more details). Collectively, these results suggest that the meta-LFICS has the advantage of high stability compared to the detection by optical markers.

4. CONCLUSION In summary, we have demonstrated a novel solution for stable and quantitative detection of S. aureus with ng/mL range of detection limit by integrating metamaterial and microfluidic technology with the LFICS. The LOD and sensitivity of the meta-LFICS reaches 0.784 ng/mL and 10.214 MHz (ng/mL), respectively. The LOD level is about 63 times higher than that of the color-based strip determined by naked eye (50 ng/mL), and the stability of the proposed strip is about 18 times higher than that of the fluorescent strip by optical microscope. In particular, this stable and comprehensive detection method overcomes the annoying interference in optical methods such as self-absorption, specular reflection, and long-term signal quenching. This work 23 ACS Paragon Plus Environment


could not only provide a powerful diagnosis tool for the quantitative detection of S. aureus or other molecules, but also deliver new avenues for achieving electric field detection of biomoleculars on membranes, system-level integration of biosensors, and the development of portable diagnostic devices.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge. Reagents and materials; apparatus; preparation of nucleic acid solution of S. aureus; recombinase aided amplification of S. aureus nucleic acid; channel width before and after baking; fabrication process of the hydrophilic channels and hydrophobic barriers; fabrication process of the SSR resonator; experimental setup for the meta-LFICS; specificity analysis of the meta-LFICS; shelf life evaluation of the meta-LFICS; matrix effects in the meta-LFICS; comparison of different test methods. (PDF)

AUTHOR INFORMATION Corresponding Author * Email: [email protected] (X. J. Mu) * Email: [email protected] (Y. Yang) * Email: [email protected] (M. Chen) ORCID Xiaojing Mu: 0000-0003-2024-2595 Ya Yang: 0000-0003-0168-2974 24 ACS Paragon Plus Environment

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Author Contributions Hong Zhou and Cheng Yang contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project was supported by National Natural Science Foundation of China (Grant No. 51605060, No.51472055, No. 81430053), Fundamental Research Funds for the Central Universities (No. 2018CDPTCG0001-5), National Key Research and Development Program of China (Grant No.2016YFB0402702, No.2016YFA0202701), and Chongqing basic science and frontier technology research project (cstc2017jcyjAX0237).

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Figure 1. Schematic illustration of (a) the meta-LFICS integrated with the SSR resonator and the microchannel. Inset: the lateral flow component for maintaining immunochromatography, including sample pad, conjugate pad, test line, control line and absorbent pad. (b) Electric field and surface current distribution of SSR resonator obtained by using the finite element simulation software ANSYS HFSS. (c) The sensitive part of the SSR resonator with split capacitance (Cs). (d) The equivalent circuit of the SSR resonator involving Rs, Ls, Cs and ∆C, after the target analyte was attached to the split of the SSR resonator. (e) The corresponding shift of the transmission spectra, when there is a change (∆C) in split capacitance (Cs). 162x67mm (300 x 300 DPI)

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Figure 2. Fabrication procedures of the meta-LFICS. (a)-(b) Hydrophilic channel and hydrophobic barrier were formed by wax-printed technology. (c) The SSR resonator was integrated onto the printed membrane by magnetron sputtering. (d) T-line and C-line were drawn in hydrophilic channel by a dispenser. (e) The biomolecule adsorbed membrane was cut into sensor elements for the subsequent preparation of LFICS. (f)(g) Sample pad, conjugate pad, absorbent pad and backing plate were assembled together into the metaLFICS. (h) Photograph of the meta-LFICS. Upper panel: the dimension of the resonator and membrane. (a1)-(c1) Cross-section optical images of strip in step (a)-(c), Insets: the corresponding enlarged images. (a2)-(c2) Schematic diagram of corresponding cross-section when filled with aqueous solution. 161x84mm (300 x 300 DPI)



Figure 3. Characterization of the hydrophilic channel and the SSR resonator. SEM images of the (a, d) bare membrane, (b, e) wax covered membrane, and (c, f) Au covered membrane. (g) AFM micrograph showing details of the hydrophilic channel. (h) The CA of wax covered membrane. (i) The optical micrograph of the split of the SSR resonator. 165x116mm (300 x 300 DPI)


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Figure 4. The proof-of-concept demonstration of biomolecules sensing using bare meta-LFICS. (a) The model, simulated, and measured transmission spectra of the empty strip. (b) Resonant frequency of strips 1# to 9#. Inset: Top view of the strip without any immobilization of biomolecules. (c) The measured transmission spectra of strips 1# to 9# when various concentrations of glucose are loaded into the channel. (d) The corresponding resonant frequency shift for glucose with a fitting curve of y = 13.2664 x + 8.3763. Inset: the schematic diagram of detecting glucose molecules using the proposed strip without any immobilization of biomolecules. 163x122mm (300 x 300 DPI)



Figure 5. Detection mechanism of S. aureus using the proposed meta-LFICS. Biological reaction in the lateral flow component when (a) positive and (b) negative sample were added to the meta-LFICS. Schematic diagram of molecular detection by the SSR resonator when the nucleic acid of S. aureus is (c) present or (d) absent on the test line. Inset: Corresponding frequency shift. Photograph of the meta-LFICS after (e) positive and (f) negative sample were loaded. 155x125mm (300 x 300 DPI)


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Figure 6. The measured results of S. aureus using the proposed meta-LFICS. (a) The measured fluorescence intensity during recombinase aided amplification of nucleic acid. (b) The measured frequency shifts of the strips when the concentrations of S. aureus vary from 0 ng/mL to 100 ng/mL. The error bar plots are extracted from 4 different samples for each concentration, and the insets are the photo of T-lines for strips at various concentrations. (c) Fluorescence images of the T-line and C-line at various concentrations. (d) Comparison of the stability between the fluorescence intensity from the fluorescent strip and the frequency shift from our meta-LFICS. 156x107mm (300 x 300 DPI)



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Integrating Microwave Resonator and Microchannel with

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