Enhancing the Sensitivity of Lateral Flow Immunoassay by

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Enhancing the sensitivity of lateral flow immunoassay by centrifugation-assisted flow control Minjie Shen, Yiqi Chen, Yunzeng Zhu, Mangsuo Zhao, and Youchun Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00421 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Analytical Chemistry

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Enhancing the sensitivity of lateral flow immunoassay by

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centrifugation-assisted flow control

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Minjie Shen, a Yiqi Chen, a Yunzeng Zhu, a Mangsuo Zhao, b and Youchun Xu a, c*

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a

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China;

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b

Tsinghua University Yuquan Hospital, Beijing 100049, China;

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c

National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.

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*Correspondence should be addressed to Y.X. ([email protected]).

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Tel: (86)-10-62796071.

Department of Biomedical Engineering, Tsinghua University School of Medicine, Beijing 100084,

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ACS Paragon Plus Environment

Analytical Chemistry 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

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Abstract

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Lateral flow immunoassay (LFIA) is widely used but is limited by its sensitivity. In this

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study, a novel centrifugation-assisted lateral flow immunoassay (CLFIA) was proposed that

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had enhanced sensitivity compared to traditional LFIA based on test strips. For CLFIA, a

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vaulted piece of nitrocellulose membrane was prepared and inserted into a centrifugal disc.

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Powered by the centrifugal force, the sample volume on the disc was not limited and the flow

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rate of the reaction fluid was steady and adjustable at different rotation speeds. It was found

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that lower rotation speeds and larger sample volumes resulted in greater signal intensity in the

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nitrocellulose membrane as well as higher sensitivity, indicating that the actively controlled

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flow on the disc allowed for sensitivity enhancement of CLFIA. To operate CLFIA on the

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centrifugal disc, a portable and cost-effective operating device was constructed to rotate the

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disc with a stepper motor and collect the results with a smartphone. The proposed method was

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successfully applied to detect prostate specific antigen (PSA) in human serum. Standard curves

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were established for CLFIA and LFIA, and both had correlation coefficients of up to 0.99.

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Under optimal conditions (1500 rpm rotation speed, 120 μL sample volume), the detection limit

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of CLFIA reached 0.067 ng/mL, showing a 6.2-fold improvement in sensitivity compared to

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that of LFIA. With clinical serum samples, a good correlation was observed between PSA

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concentrations measured by CLFIA and by a bulky commercial instrument in hospital. In

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summary, this portable, cost effective, and easy-to-use system holds great promise for

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biomarker detection with enhanced sensitivity compared to traditional LFIA.

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Introduction

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Lateral flow immunoassay (LFIA) is one of the most popular methods for point-of-care

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testing (POCT).1,2 The vast majority of LFIA is performed in the form of lateral flow strips

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(LFS). For decades, LFS have been manufactured and widely applied to healthcare, food safety,

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and environmental monitoring. Specific applications include pregnancy tests, infectious

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diseases,3-6 inflammation,7,8 disease biomarkers,9-12 foodborne pathogens,13,14 toxins,15-17

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pesticides,18 and drug residues.19,20 LFS have gained a great deal of interest and profit due to

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the major advantages including their ease of use, rapid response, low cost, and long shelf life.

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However, the analytical performance of traditional LFIA is still limited by its sensitivity.

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First, to overcome this limitation, alternative materials have been used in place of traditional

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gold nanoparticles (AuNPs) or to apply signal amplification. For instance, carbon

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nanoparticles/nanotubes,21,22

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materials,10 and magnetic nanoparticles26,27 have been reported as labels resulting in improved

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LFIA sensitivity. In addition, silver and gold enhancement methods28 and enzyme/nanozyme-

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based colorimetric amplification methods29,30 have also been exploited in LFIA. Although these

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novel materials provide new approaches for highly sensitive detection, additional instruments

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are more or less required for quantitative measurements due to specific optical, magnetic or

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electrochemical properties of these materials. Besides, for the signal amplification methods,

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additional assays will be needed which might otherwise make the corresponding instruments

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for automated detection cumbersome. Second, in addition to advancements in signal

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presentation, other characteristics of LFIA related to sensitivity have also been explored. Parolo

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et al. altered the architecture of test strips, achieving a significant improvement in sensitivity.31

fluorescent

materials,23

quantum

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dots,24,25

upconverting

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Rivas et al. constructed wax pillar modified strips, which caused microfluidic delay and

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generated pseudoturbulence on the detection membrane, resulting in better performance.32

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These improvements indicate another strategy to enhance LFIA’s sensitivity by changing the

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sample flow on the strip. However, the change of the flow in these studies31,32 is not actively

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adjustable. Third, the performance of LFIA is also constrained by intrinsic defects. A lateral

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flow test strip basically consists of a sample pad, a conjugate pad, a nitrocellulose (NC)

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membrane, and an absorbent pad. The immobilization of reaction reagents on a strip is

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restrained by the capacity of these compartments as well as the sample loading volume.

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Moreover, liquid propulsion by capillary forces cannot be precisely controlled, which may

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generate different liquid flow rates owing to variations in sample viscosity and surface

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tension.33 Integrating the LFIA into centrifugal microfluidic platforms34 with active flow

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control may address the aforementioned issues.

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Over the past decade, we have been continuously working on the development of

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microfluidic devices for biomedical-related applications,35,36 and recently dedicated in

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innovation and development of centrifugal microfluidic platforms.37,38 Unlike using LFS as a

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unit for final detection on microfluidic platforms,14,39 we herein present a controlled LFIA in

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centrifugal discs, named centrifugation-assisted lateral flow immunoassay (CLFIA), for the

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first time. In CLFIA, a vaulted piece of NC membrane, with a test and a control line, was

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prepared and inserted into a centrifugal microfluidic disc. The AuNP-labeled antibody was

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lyophilized and preloaded into the disc. Different volumes of sample can be loaded into the disc

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in CLFIA without the limitation accompanied by the capacity of the strip in LFIA. The liquid

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was driven through the NC membrane by the cooperation of the centrifugal and capillary forces. 5 / 28



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Meanwhile, the liquid flow rate was much more stable than that of LFIA and could be adjusted

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under different rotation speeds of the disc. Combining these merits, the improved sensitivity of

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CLFIA was demonstrated compared to the traditional LFIA. To support the operation of this

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centrifugal disc, a portable device was constructed to rotate the disc and capture the results

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using a smartphone. To further assess our method, human prostate specific antigen (PSA) was

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chosen as the target to validate the improved performance of our method.

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Experimental section

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Preparation of AuNP-labeled antibody

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The AuNP-labeled antibody was prepared according to the protocol described previously.40

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Briefly, incubation buffer was prepared by adding 3.09 g boric acid (Sinopharm Chemical

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Reagent, Shanghai, China) into 500 mL ultra-pure water from the Milli-Q water purification

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system (Millipore, Beijing, China), and the pH was adjusted to 8.0. Then, a volume of 1.5 mL

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gold solution (20 nm, OD520 = 0.10, Beijing Kwinbon Biotechnology, Beijing, China) was

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centrifuged at 10000 × g for 20 min and the buffer was exchanged with 0.8 mL incubation

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buffer. An appropriate amount of conjugate antibody (10-3143, Fitzgerald, MA, USA) was

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added into the gold solution and mixed using a roller mixer at 25°C. After incubation for 1 h,

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incubation buffer (200 μL) containing 10% bovine serum albumin (BSA, Sigma-Aldrich,

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Shanghai, China) was added into the gold solution and incubated for another 20 min to block

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any unconjugated sites on the AuNPs. The solution was centrifuged at 10000 × g for 20 min at

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4°C and the supernatant was discarded. The pellet was resuspended in 0.5 mL storage buffer

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(incubation buffer containing 1% BSA) to eliminate any unconjugated antibody. Subsequently,

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the solution was centrifuged again (10000 × g, 20 min, 4°C). The pellet was finally resuspended

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in 200 μL storage buffer and stored at 4°C until use. After adding 10% trehalose (Sigma-Aldrich,

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Shanghai, China), a portion of the final solution was lyophilized into small globules for use on

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the disc.

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Preparation of LFS

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The NC membrane (HF13502S25, Millipore, Beijing, China), sample pad, and absorbent

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pad (Beijing Kwinbon Biotechnology, Beijing, China) were incorporated onto a plastic backing 7 / 28



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card successively. Capture antibody (2 mg/mL, 10-3142, Fitzgerald, MA, USA) and anti-mouse

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IgG (1 mg/mL, ab7063, Abcam, Shanghai, China) were dispensed onto the NC membrane as

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the test (T) and control (C) lines, respectively, using a dispenser (XYZ3050, BioDot, Shanghai,

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China). The dispensing rate was 0.5 μL/cm. The AuNP-labeled antibody solution was dispensed

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onto the conjugate pad using an IsoFlow dispenser (Imagene Technology, NH, USA) at the rate

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of 16 μL/cm. The fabricated pad was then dried at 37°C for 10 h, cut into 3 mm wide strips,

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and stored at room temperature in a desiccator.

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Fabrication of CLFIA discs

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The CLFIA disc was made of polymethyl methacrylate (PMMA) and engraved by

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Hongyang Laser Co. Ltd. (Beijing, China). The lyophilized AuNP-labeled antibody was loaded

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into the loading chamber on the disc. The lyophilizing process is only to facilitate the long-term

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storage and easy use of the disc, and users can also directly load the buffer with AuNPs-labeled

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antibody for disc using. The NC membrane for the disc was prepared using the same procedure

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as described for LFS, except that the NC membrane was cut into the designed pattern (Fig. 1a)

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using a cutting plotter (FC4500-50, Graphtec, Yokohama, Japan) with a width of 3 mm. The

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NC membranes cut for LFS and CLFIA disc have similar total area (about 70 mm2). The back

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of the patterned NC compartment was attached to an adhesive tape (Youbisheng Adhesive

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Products, Hangzhou, China) and the tape was then stuck to the PMMA disc to seal the chambers

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and channels. The NC compartment was aligned to the NC chamber when attaching the tape to

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the disc.

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Design of the operating device for CLFIA

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The shell of the device was fabricated by 3D printing (WeNext Corp., Shenzhen, China). A 8 / 28


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stepper motor (YZ-ACSD608, Yizhi Technology, Shenzhen, China) was attached to the bottom

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of the device. A 3D printed pallet, on which the disc was set, was fixed to the spindle of the

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motor. A photogate (Panasonic, Shanghai, China) and the positioning hole of the pallet were

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used to stop the disc at the specific angle for final signal acquisition. For illumination, two flat

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LED light sources (Baohui optoelectronics, Shenzhen, China) were attached to the

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photographic chamber. A smartphone (Mi 4, Xiaomi, Beijing, China) was positioned over the

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apparatus to photograph the results. The schematic of the device is shown in Fig. S1. The device

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was powered by an external power supply and controlled by a computer.

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Performance evaluation of liquid manipulation

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The working buffer was 10 mM phosphate buffer (PBS, pH 7.4, Solarbio, Beijing, China)

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containing 0.2% (V/V) Tween-20 (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China).

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First, the performance of liquid migration on LFS and on CLFIA discs was evaluated. For LFS

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evaluation, the working buffer (120 μL) was transferred into a 1.5 mL centrifuge tube. Then,

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the strip was added to the tube and the remaining volume of buffer was quantified at different

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incubation times. For disc evaluation, the working buffer (120 μL) was transferred into the

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loading chamber and centrifuged at a defined rotation speed (2000 rpm). Images were taken at

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the different time points, and the remaining volume of buffer in the reaction chamber was

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calculated by analyzing each picture (Fig. S2). Second, the flow rates of fluid through the NC

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membrane on the disc were also measured at different rotation speeds. The same amount of

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working buffer (60 μL) was loaded and centrifuged at 1250, 1500, 2000, 2500, or 2750 rpm.

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The time required for all of the liquid to transfer from the reaction chamber to the waste

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chamber at each rotation speed was recorded. 9 / 28



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Immunoassay

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PSA standard solutions at different concentrations were prepared by diluting a PSA stock

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solution (30C-CP1017U, Fitzgerald, MA, USA) in the working buffer for performance

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evaluation. First, with a fixed rotation speed, signals obtained with an increasing volume of

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solution were measured on both LFS and CLFIA discs. Second, with a specified volume of

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PSA standard solution, signals at different rotation speeds were measured on CLFIA discs.

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Third, at a fixed reaction time, the signal intensity at different rotation speeds with a sufficient

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volume of PSA standard solution were measured on CLFIA discs. For serum tests, PSA spiked

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serum samples and clinical serum samples were analyzed on both platforms. Tests on LFS were

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completed in 15 min. Tests on discs were performed at corresponding optimal conditions. All

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experiments were performed at room temperature. Data were collected using a smartphone and

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quantitative analysis was performed on a computer using the Image J software.

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The study was approved by the Institutional Review Board (IRB) of Tsinghua University

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of China (No. 20180022). All serum samples were provided by donors following IRB

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guidelines.

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Results and discussion

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Principle of the CLFIA system

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The details of the patterned NC membrane and the fabrication of a disc are shown in Fig.

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1a-b and Fig. S3. For disc use, the sample is injected into the loading chamber (Fig. 1c). The

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lyophilized AuNP-labeled antibody will be instantly dissolved. Then, the disc is rotated at a

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defined speed. The NC membrane for CLFIA has a bridge-like shape and guides the liquid from

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the upper chamber to the lower. After the reaction liquid transfers to the reaction chamber and

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contacts the membrane, the liquid on the left of the NC bridge will be dragged by centrifugal

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and capillary forces, which are in opposite directions. At rotation speeds below 3000 rpm, the

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capillary force prevails, and the liquid will move forward along the NC bridge. The vaulted

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structure of the NC bridge can lead the liquid to the right where the liquid will be continuously

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impelled from the membrane to the waste chamber, forming a constant capillary force for the

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left hand side of the NC bridge. Therefore, the sample is continuously transferred through the

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NC bridge and the immunoassay steadily proceeds. For the immunoassay to detect PSA, the

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antigen in the sample conjugates to the AuNP-labeled antibody and is then captured by another

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antibody on the test line. The redundant AuNP-labeled antibody not bound to antigen will move

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forward and be captured by the antibody on the control line. Thus, color signals can be

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generated on the test and control lines. The gray intensity of the test line is obtained for

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quantitative analysis. The schematic of LFIA was shown in Fig. S4. A brief video showing the

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detection process is available in the Supporting Information.

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The vaulted NC membrane in CLFIA system is a deliberate design inspired by the structure

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of the siphon valve wildly used on centrifugal platform.41 With this bridge-like shape, the liquid 11 / 28



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transferring from upstream to downstream chambers is steady and adjustable. Additionally, the

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angle between the two hands of the NC membrane was definite. The two hands were located in

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a radial pattern so that it is coincident with the direction of centrifugation force, facilitating the

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uniformity of the lateral flow on the membrane.

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The CLFIA system provides different approaches for the enhancement of LFIA, which are

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mainly manifested in two aspects. First, sufficient time period is needed for the formation of

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immune-complex at the test and control lines to ensure firm binding of the reagents at molecular

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level. The flow rate is a determining factor for this time period. Second, with a certain flow

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rate, sample volume determines the whole reaction time at the test and control lines. Longer

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reaction time results in more immune-complexes at the reaction lines until saturate. Therefore,

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the control of the flow rate and the adjustment of the sample volume may aid to improve the

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sensitivity of LFIA.

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Performance of liquid manipulation

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The liquid on LFS is driven passively by the capillary force generated by the absorbent pad.

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Once the process starts, the liquid movement is determined by the strip itself. Conversely, the

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liquid on the NC bridge that inserted into the CLFIA disc is actively controlled by the

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centrifugal and capillary forces acting on it. As shown in Fig. 2a, the liquid volume on LFS

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increases rapidly at the beginning and then gradually a slow increase is observed until the liquid

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volume reaches the maximum the strip can contain. The liquid on LFS at the beginning is

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absorbed by the sample pad and conjugate pad. Therefore, the effective volume through the NC

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membrane on LFS is less than 40 μL during the entire process. Whereas, the liquid volume

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passing through the NC bridge in the CLFIA disc steadily increases over time without such 12 / 28


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limitation. In addition, the slope at each point on the curves in Fig. 2a indicates that the liquid

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flow rate on LFS decreased during strip tests while the flow rate through the NC bridge was

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comparatively consistent under a constant rotation speed. In addition to consistent control, the

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liquid flow rate can also be adjusted by altering the rotation speed of the CLFIA disc. As shown

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in Fig. 2b, the flow rate on the CLFIA disc increased as the rotation speed increased from 1250

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rpm to 2500 rpm. When the speed was higher than 2500 rpm, the flow rate decreases. Here, the

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adjustable range of rotation speed is constrained between 1250 rpm to 2750 rpm to ensure the

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lateral flow works on the disc. When the rotation speed is below than 1250 rpm, the liquid at

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the right hand side of the NC bridge is retained by the membrane because the capillary force is

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higher than the centrifugal force that tends to drive the liquid out of the NC bridge. When the

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rotation speed is higher than 2750 rpm, the centrifugal force can impede the liquid at the left

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hand side of the NC bridge to flow upward. These results show the overwhelming fluid control

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capability of the CLFIA system over that of LFS. It demonstrates that the sample volume and

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liquid flow rate through NC membrane are no longer non-adjustable on CLFIA discs.

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Performance evaluation of CLFIA at adjustable conditions

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There are plenty of factors that can affect the performance of CLFIA, which can be

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generally divided into two groups determined by the biochemical characters of reaction system

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and the spinning program, respectively. The first group includes the composition and viscosity

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of the sample, the quality of AuNPs, and the specificity of the antibodies. The second group

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includes the rotation speed and the reaction time period. Since the first group of factors is

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determined by the characteristics of the testing object and the commercial reagents, there is

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little possibility for us to make comprehensive optimization. Only the size, modification and 13 / 28



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dosage of AuNPs were examined. For the size of AuNPs, it is reported that AuNPs with

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comparatively large diameter benefit to produce stronger signals42,43 while they are more likely

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to sediment under centrifugation. Therefore, AuNPs at diameter of 20 nm were chosen to

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balance these two effects. For the modification of AuNPs, different dosages of antibody were

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added for AuNPs labeling as shown in Fig. S5, and the result showed that 5 μg antibody was

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sufficient for labeling. In addition, the dosage of AuNP-labeled antibody used for LFIA was

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also optimized (Fig. S6), allowing the calculation of the amount of AuNP-labeled antibody

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deposited on the LFS conjugate pad and CLFIA disc.

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As detailed in Fig. 2a-b, liquid volume and flow rates on CLFIA are adjustable, therefore,

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we mainly focus on the second group of factors to improve the sensitively of immunoassay.

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Fig. 3a shows the signal intensity as a function of sample volume in LFIA and CLFIA. As the

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sample volume increased, signal of LFIA increased until it reached the maximum determined

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by the capacity of the strip. Signal of CLFIA increased along with the sample volume. At the

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same sample volume, the signal of CLFIA was much stronger than that of LFIA, because the

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sample could be fully utilized in CLFIA but the sample on the strips might remain in the

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sample pad or conjugate pad or evaporate. Another reason for poor signal of LFIA is that the

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majority of the AuNP-labeled antibody is flushed out at the onset of testing, causing the

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inefficient reaction of the remaining sample. As for performance evaluation at different flow

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rates, signal intensity using equal volumes of sample at different rotation speeds was measured

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(Fig. 3b). As the rotation speed increased from 1500 rpm to 2500 rpm, the flow rate increased

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and the signal intensity decreased, indicating that increased flow rate reduced the reaction

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efficiency between the antibodies and antigen. This is because the exposure time is reduced at 14 / 28


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higher flow rates. However, the further reducing of the rotation speed (1250 rpm) may not be

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helpful for signal enhancement because the molecules can get adequate time to contact and

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bind to each other when the flow rate is low enough. As shown in Fig. 3b, the signals have no

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significant difference when the rotation speed is below than 1500 rpm. At the rotation speed

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of 2750 rpm, the AuNP-labeled antibody starts to precipitate, which affects the signal. Once

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the reaction time of CLFIA is set, the signal will be a compromise between the sample volume

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and flow rate at one rotation speed. Fig 3c shows that 2000 rpm is a turning point with

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comparatively high signal intensity for the same reaction time as LFIA. To sum up, these

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results indicate that CLFIA allows much greater flexibility in adjusting sample volume and

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flow rate, which will be important parameters for CLFIA system to optimize the detection

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signal.

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Since the sensitivity and the operating period are two factors that should take into

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consideration but have different importance in different applications, two optimal conditions

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can be proposed for quick tests with acceptable sensitivity and sensitive tests with acceptable

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duration, respectively. For quick tests (15 min), the optimal rotation speed is 2000 rpm, as

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shown in Fig. 3c. The consumed sample in this condition (condition 1) is about 60 μL in

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volume. For sensitive tests with acceptable duration (45 min), lower rotation speed (1500 rpm)

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is facilitate to obtain stronger signal, as shown in Fig. 3b. The consumed sample in this

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condition (condition 2) is about 120 μL in volume.

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Performance validation of CLFIA using PSA spiked serum samples

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Serum samples from five healthy females were used as control samples and to prepare PSA

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spiked serum samples, as the PSA concentration in healthy female serum is far below the 15 / 28



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detection capability of LFIA. It was further confirmed because the detected results of control

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samples in CLFIA system remained low and stable without any nonspecific signal at increasing

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sample volumes (Fig. S7). PSA spiked serum samples in the concentration range of 0-10 ng/mL

275

were analyzed both on LFS and CLFIA discs with optimized condition 1 and 2. Each sample

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was diluted 4 times with the working buffer before each assay was performed. Despite the

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optimization factors of the reaction system mostly determined by the characteristics of the

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sample and commercial reagents, condition 2 is the most optimal among all the conditions

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within adjustable range unless using a new disc with larger sample capacity.

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Visualized with the naked eye, the color intensity of the T line increased as the PSA

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concentration increased. The PSA concentrations that could be explicitly distinguished by LFIA,

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CLFIA condition 1, and CLFIA condition 2 were 5 ng/mL, 1 ng/mL, and 1 ng/mL, respectively

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(Fig. 4a-c). Using a smartphone to capture images of LFS and CLFIA discs, quantitative

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analysis was performed using Image J. Standard curves are shown in Fig. 4d-f and the curve-

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fitting equations and logistic correlation coefficients (R2) are also shown in the figures. The

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limit of detections (LODs) of LFIA, CLFIA condition 1, and CLFIA condition 2 reached 0.41

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ng/mL, 0.13 ng/mL, and 0.067 ng/mL, respectively. The R2 values were all above 0.99,

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indicating a good correlation between the concentration of PSA and ΔGray intensity. The

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reproducibility of the measurements for PSA in serum by different methods was also studied

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and the relative standard deviations (RSD) were shown in Table 1. Lower RSD values at each

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PSA concentration reflected better reproducibility of CLFIA than that of LFIA. We further

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evaluated the specificity of our system by testing three proteins (alpha fetoprotein,

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carcinoembryonic antigen, human chorionic gonadotropin) and a good specificity was obtained 16 / 28


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as shown in Fig. S8. Besides, it is possible to insert more units on a disc for repeatable tests or

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multiple detections, possessing the potential for better efficiency and reproducibility.

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The results clearly demonstrate that CLFIA has the capability to detect PSA at lower

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concentrations than those detected by LFIA. In addition, CLFIA has the potential to detect even

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lower concentrations of the target antigen using larger sample volumes. Compared to LFIA,

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CLFIAs at the two tested conditions had better sensitivity, with an LOD improvement of up to

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3.2-fold and 6.2-fold, respectively. Moreover, our method can be adjusted according to the

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operation conditions, which can greatly increase its applicability.

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Compared to the previous PSA detection methods (Table S1), our method utilizes the most

303

basic materials with an affordable cost and has a decent performance at the same time. In

304

addition, our method has inherent advantages for POCT applications due to its high automation

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and quick response as well as a portable supporting device. More importantly, the

306

centrifugation-based strategy is compatible with many sensitivity enhancing methods reported

307

before to achieve more optimized results.

308

Determination of PSA in clinical serum samples

309

Analytical performance of CLFIA was tested with clinical serum samples. Serum samples

310

were diluted 4 times with the working buffer, and the PSA concentration in each sample was

311

determined by CLFIA under condition 2. PSA concentrations were calculated according to the

312

standard curve (Fig. 4f). Each sample result was plotted in Fig. 5. The regression coefficient of

313

the equation is close to 1 and the R2 value is above 0.97, indicating a good agreement between

314

the PSA concentrations measured by CLFIA and by electrochemiluminescence (ECL) with a

315

commercial bulky instrument in a hospital. Besides, compared with previous researches,7-10 the 17 / 28



316

relative deviations between CLFIA and ECL results were all within tolerance considering the

317

variability of serum samples (Table S2), indicating a good accuracy. The results demonstrate

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that our method possesses the potential for clinical analysis. Moreover, our method can

319

additionally provide valuable monitoring data at low biomarker concentrations compared to

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traditional LIFA (Fig. 4).

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321


Conclusion

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A novel centrifugation-assisted lateral flow immunoassay was developed for the first time

323

by inserting a piece of NC membrane into a centrifugal microfluidic disc. Powered by the

324

centrifugal force, the process of immunoassay was actively controlled. The sample volume and

325

liquid flow rate through the NC membrane, associated with the sensitivity, can be easily

326

adjusted. Under optimal conditions, our method showed 6.2-fold improvement in the LOD for

327

PSA measurements in serum compared to traditional LFIA by test strips. Additionally,

328

multistep operations including sample preparation and downstream amplification are readily

329

integrated, owing to the advantages of the centrifugal microfluidic platform. The centrifugal

330

immunoassay system proposed in this study provide a universal strategy that can be applied for

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many analytes based on affinity interaction. Assisted by a portable and cost-effective

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smartphone-based automatic operating device, this novel immunoassay method with increased

333

sensitivity shows great potential in healthcare, food safety, environmental monitoring, and

334

several other complex applications.

335

336

Acknowledgments

337

This study was supported by the National Key Research and Development Program of

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China (2016YFC0800703), the National Natural Science Foundation of China (31870853), and

339

the Beijing Lab Foundation.

340

341 342

Supporting Information Schematic of the operation device of CLFIA; volume measurement on captured images; 19 / 28



343

manufactural schematic of the vaulted NC membrane; schematic of LFIA; optimization of

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AuNP labelling and the dosage of AuNP-labeled antibody; specificity test; summary of

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different methods for PSA detection; relative deviations between CLFIA and ECL results.

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References

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420 421

Fig. 1 Schematic of the CLFIA system. (a) The patterned NC membrane. T and C show the

422

positions of the test and control lines, respectively. (b) The configuration of the CLFIA disc.

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(c) Illustration of the patterned PMMA layer. (d) An assembled CLFIA disc. (e) The

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smartphone-based operating device.

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425 426

Fig. 2 Performance of liquid manipulation. (a) Comparison of the liquid volume on LFS and

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through the NC membrane on CLFIA discs at different reaction times. (b) Liquid flow rates

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through the NC membrane of CLFIA at different rotation speeds. The curves in this figure are

429

fitted as guides to the eye. Error bars show standard deviations of triplicate measurements.

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430 431

Fig. 3 Signal intensities at different conditions. (a) Signal intensity using different volumes of

432

PSA standard solution at a concentration of 3 ng/mL and rotation speed of 2000 rpm.

433

Photographic images of (i) LFIA results and (ii) CLFIA results with different sample volumes.

434

(iii) Quantitative comparison of LFIA and CLFIA results. “Blank” means the background using

435

120 μL working buffer without PSA. (b) Signal intensity of CLFIA at different rotation speeds

436

using PSA standard solution (60 μL) at a concentration of 1 ng/mL. (i) Photographic images of

437

CLFIA results and (ii) quantitative signal analysis at different rotation speeds. (c) Signal

438

intensity of CLFIA at different rotation speeds for 15 min with sufficient PSA standard solution

439

(1 ng/mL). (i) Photographic images of CLFIA results and (ii) quantitative signal analysis at

440

each rotation speed. ΔGray intensity = signal intensity - background intensity. Error bars show

441

standard deviations of triplicate measurements.

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442 443

Fig. 4 Detection results with PSA spiked serum samples. (a-c) Photographic images of LFIA,

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CLFIA condition 1, and CLFIA condition 2, respectively, with PSA spiked samples at different

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concentrations. (d-f) Calibration curves for LFIA, CLFIA condition 1, and CLFIA condition 2,

446

respectively. Error bars show standard deviations of triplicate measurements.

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Table 1 Relative standard deviations of results at different PSA concentrations measured by

448

LFS, CLFIA condition 1, and CLFIA condition 2. (n = 3)

449

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450 451

Fig. 5 Correlation between PSA concentrations of clinical serum samples measured by CLFIA

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and by ECL.

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Enhancing the Sensitivity of Lateral Flow Immunoassay by

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