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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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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Analytical Chemistry

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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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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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Analytical Chemistry

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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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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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Analytical Chemistry

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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

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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

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

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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

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

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before to achieve more optimized results.

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Determination of PSA in clinical serum samples

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

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

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

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

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

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

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

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

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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

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additionally provide valuable monitoring data at low biomarker concentrations compared to

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traditional LIFA (Fig. 4).

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Conclusion

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

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

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

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

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

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

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multistep operations including sample preparation and downstream amplification are readily

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

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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

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

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several other complex applications.

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Acknowledgments

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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

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the Beijing Lab Foundation.

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341 342

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

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

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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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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

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

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

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

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

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

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

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

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

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

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

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

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standard deviations of triplicate measurements.

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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,

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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

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

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

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and by ECL.

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