Label-Free Photoelectrochemical Immunosensor for Neutrophil

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A label-free photoelectrochemical immunosensor for neutrophil gelatinase-associated lipocalin based on the novel use of Nanobodies Henan Li, Yawen Mu, Junrong Yan, Dongmei Cui, Weijun Ou, Yakun Wan, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504589d • Publication Date (Web): 04 Jan 2015 Downloaded from http://pubs.acs.org on January 13, 2015

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Analytical Chemistry

A label-free photoelectrochemical immunosensor for neutrophil gelatinase-associated

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lipocalin based on the novel use of Nanobodies

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Henan Li,‡a Yawen Mu,‡b Junrong Yan,b Dongmei Cui,a Weijun Ou,c Yakun Wan,b,c*

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Songqin Liu,a*

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a

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China.

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b

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of Life Sciences, Southeast University, Nanjing 210000, P.R. China.

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c

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*

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Nanjing 211189, P. R. China. Tel.: 86-25-52090613; Fax: 86-25-52090618.

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E-mail addresses: [email protected] (S. Q. Liu)

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Institute of Life Sciences, Southeast University, Nanjing 210096, P.R. China. Tel.: 86-25-83790967;

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Fax: 86-25-83790960.

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E-mail addresses: [email protected] (Y. Wan)

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‡ These authors have equally contributed to this work.

School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, P.R.

The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute

Jiangsu Nanobody Engineering and Research Center, Nantong 226010, P.R. China. Corresponding author at: School of Chemistry and Chemical Engineering, Southeast University,

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ACS Paragon Plus Environment


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ABSTRACT:

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Acute renal failure (ARF) represents a very important and potentially devastating disorder in

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clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for

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ARF in a wide range of different disease processes, which is frequently detected in clinical

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diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC)

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immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly

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immobilized to streptavidin-coated 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2

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electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the

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antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC

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immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of

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the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated

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CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead

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to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been

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significantly lowered to 0.6 pg mL-1. This proposed immunosensor reveals high specificity to

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detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the

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present work provides a new approach to design Nb-based PEC immunosensor and increases

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versatility of Nbs.

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Introduction

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Acute renal failure (ARF) remains a common and potentially devastating disorder in clinical

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nephrology, with a persistently high mortality and morbidity rate despite significant advances in

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supportive care.1, 2 Many pioneering studies have highlighted the pathogenesis of ARF and have

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paved the way for successful interventions in animal models, but translational research efforts in

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patients have yielded disappointing results due to the scarcity of early detection of biomarkers for

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ARF with an unacceptable delay in initiating therapy.3 Advances in basic science research have

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identified that neutrophil gelatinase-associated lipocalin (NGAL) is a highly sensitive, specific,

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and predictive early biomarker for ARF in a wide range of different disease processes.4 Therefore,

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developing rapid and sensitive detection methods for NGAL can promote the ability to launch

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preventive and therapeutic measures for this disorder in a timely manner. There are several

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conventional methods to detect NGAL, such as enzyme-linked immunosorbent assay (ELISA),5

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chemiluminescence assay,6 and localized surface plasmon resonance (LSPR).7

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strategy has distinct advantages, yet suffers significantly from limited sensitivity and high

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equipment cost. Therefore, accurate, ultrasensitive, label-free and low-cost NGAL detection is

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still of critical urgency for early ARF diagnosis.

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While every

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Photoelectrochemical (PEC) sensing approach, a new kind of detection method based on

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photoinduced electron transfer processes at electrode/solution interfaces, has received profound

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interest for its desirable high sensitivity, low background, and easy minimization.9-17 Recently,

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great efforts have been made to detect various analytes, such as immune molecules, DNA

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oligonucleotides, enzyme inhibitors, cells and some small molecules.18-25 Among those, the PEC

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label-free immunosensor is a rapid and high-throughput biological assay combing the advantages

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of label-free immunoassay and PEC method, which can avoid the process of labeling antibody or

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antigen with makers, making the experimental process simple, low cost and time saving.13, 26 The

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antigen concentrations were directly measured through the steric hindrance-induced decrease in

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photocurrent intensity resulting from the specific immunoreaction, where its specific antibody

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was immobilized on one side of the sensor as a receptor molecule.

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Nanobodies (Nbs), a distinct type of antibody fragments, is the variable domain of

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heavy-chain-only antibodies found in camelids, as known as the variable domain of heavy chain 3



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of heavy-chain only antibodies (VHH), have received a progressively growing interest in

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diagnostics.27 With a molecular weight of 12 ~ 15 kDa, Nbs are the smallest recognized

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antigen-binding antibody fragment so far.28 Furthermore, Nbs exhibit high thermal and chemical

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stability, and compared with intact full-size monoclonal antibodies (mAbs) and their fragments,

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they are more robust, better water-soluble and have a lower tendency for aggregation.29,

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Because of Nbs’ small size and long protruding complementarity determining regions 3, they are

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able to recognize epitopes that are inaccessible or cryptic for conventional antibodies.31 In

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addition, the C-terminus of Nbs is situated at the opposite site of the antigen binding region, as

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this terminus is rarely involved in binding, and provides an optimal target for modification.27 Thus,

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site-specific functional groups can be easily and selectively introduced at the C-terminus,

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allowing covalent and oriented binding with minimal loss of specificity and affinity.32

30

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Here, we report a label-free and sensitive PEC immunosensor for NGAL based on

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anti-NGAL Nb immobilization method that utilizes a biotinylated Nb (BiNb) for immobilization

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to streptavidin-functionalized surfaces. As showed in the scheme 1, a healthy bactrian camel was

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immunized with NGAL. Then, the resulting mRNA coding for the Nbs was isolated, transcribed

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to cDNA, and cloned into phagemid pMECS for phage-displayed library construction. Four

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anti-NGAL Nbs have been successfully identified by phage display technology, and all of them

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demonstrated high affinity to NGAL. Additionally, the one with the highest affinity, Nb1, was

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chosen to conjugate with biotin in vivo at its C-terminus. Furthermore, we developed a

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fascinating PEC immunosensor by using streptavidin-biotin bridge to immobilize the BiNb on the

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2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode surface for the quantitative

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detection of the NGAL. This ideal stepwise bang-edge structure achieves the ultimate purposes of

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broad absorption of solar light, reduced recombination of photogenerated electron-hole pairs, and

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high-speed transfer of the excited electrons to the electrode. This Nb-based PEC immunosensor

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exhibited high sensitivity, reproducibility, specificity, and stability.

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1

Experimental section

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Chemicals and materials

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The ITO slices were purchased from Zhuhai Kaivo Electronic Components Co., Ltd. (sheet

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resistance < 7 ohm sq-1, China). TiO2 nanoparticles were obtained from Aladdin (China). Ascorbic

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acid (AA), chitosan (CS), and glutaraldehyde (GA) (25% aqueous solution) were purchased from

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Sinopharm Chemical Reagent Co., Ltd. (China). Bovine serum albumin (BSA) was purchased

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from Sunshine Biotechnology Co., Ltd. (Nanjing, China). Human chorionic gonadotropin (HCG),

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carcino-embryonic antigen (CEA), prostate specific antigen (PSA), alpha fetoprotein (AFP),

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neutrophil gelatinase-associated lipocalin (NGAL), and streptavidin (SA) were obtained from

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Shanghai Ruiqi Biological Technology Co., Ltd. (Shanghai, China). All other reagents were of

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analytical grade and used as received. All aqueous solutions were prepared with deionized water

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(DI water, 18 MΩ cm-1, Milli-Q, Millipore). 2,9,16,23-tetraaminophthalocyanine (CoPc) was

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synthesized according to previously reported literatures.33

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Apparatus

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Absorbance determination was performed on BioRad iMarkTM (Bio-Rad, USA). Surface

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plasmon resonance imaging (SPRi) binding assay was performed on PlexArray® HT system

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(Plexera LLC, USA). Scanning electron microscopy (SEM) and energy dispersive spectrum (EDS)

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were performed using an Ultra Plus field emission scanning electron microscope (Zeiss,

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Germany). Electrochemical impedance spectroscopy (EIS) was performed on a VersaSTAT 3

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workstation (Princeton Applied Research, USA) with a three-electrode system in KCl solution

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(0.1 M) containing a K3[Fe(CN)6]/K4[Fe(CN)6] (2 mM, 1:1) mixture as redox probe, and recorded

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at an open circuit potential of 0.2 V with an amplitude of 5 mV over a frequency range of

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0.01-100 kHz. The electrode system contained a modified ITO working electrode with an area of

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0.5 cm2, a Pt wire counter electrode, and a KCl saturated calomel reference electrode (SCE).

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Photocurrent was measured on a CHI 750 workstation (Shanghai Chenhua Apparatus Corporation,

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China) with visible light irradiation, which obtained by 150 W Xe lamp (Beijing NBeT Co., Ltd.

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China) with a 420 nm cutoff filter. All experiments were carried out with a three-electrode system,

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whereas the modified ITO electrode with an area of 0.5 cm2 was used as the working electrode, a

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Pt wire and a saturated (SCE) electrode were used as the counter electrode and the reference 5



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electrode, respectively. All photoelectrochemical detection was carried out at a constant potential

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of -0.2 V (vs. a saturated SCE electrode) in PBS (pH 7.4, 0.1 M) containing AA (0.1 M), which

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was degassed by highly pure N2 for 15 min before PEC experiments and then kept over a N2

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atmosphere for the entire experimental process.

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Nanobody Biotinylation

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Genes encoding anti-NGAL Nbs were sub-cloned into plasmid pBAD17 using Nco I and

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BstE II as restriction sites, then the recombinant plasmid was co-transfected into WK6 cells with

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another plasmid pBirA. 50 µM biotin was added into the medium 30 min before adding 1mM

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IPTG to induce Nb expression. Periplasmic proteins were extracted by osmotic shock. BiNb was

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purified by a Streptavidin Mutein Matrix and eluted with 4 mM biotin solution.

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Fabrication of Photoelectrochemical Immunosensor

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Briefly, the ITO electrodes were ultrasonically cleaned in 2 M boiling KOH solution solved

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in 2-propanol for 20 min, followed by washing copiously with water and dried at 120 ºC for 2h. A

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certain amount of TiO2 powder was ultrasonically dispersed in water, and then the homogeneous

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suspension (25 µL) was dropped on a piece of ITO slice. After drying in air, the film was annealed

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at 150 ºC for 10 min. Then the ITO/TiO2 electrode was immersed in a solution of CoPc (0.1 mM)

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in DMF for 1-6 h. After drying in air, a CS solution (0.05 wt%, 25 µL) dissolved in acetic acid

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(1%) was coated on the ITO/TiO2/CoPc electrode and dried at 50 ºC followed by washing with

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aqueous NaOH solution (1 M) and DI water, respectively. A solution of GA (2.5%, 25 µL) was

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dropped on the electrode and left at room temperature for 1h. Then the electrode was rinsed with

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DI water thoroughly to remove physically adsorbed GA. 25 µL of SA solution (50 µg mL-1) was

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spread onto the resulting electrode surface at 4 ºC in a moisture atmosphere to avoid evaporation

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of solvent. After incubation for 12 h, the electrode was rinsed with PBS (pH 7.4, 10 mM) to

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remove physically adsorbed SA. The electrode was then blocked with blocking solution (25 µL)

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for 30 min to block non-specific binding sites and washed with the PBS thoroughly. Then, 25 µL

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of BiNb solution (50 µg mL-1) were dropped onto the modified electrode for an incubation of 1 h

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at 37 ºC followed by washing with PBS. The ITO/TiO2/CoPc/CS/SA/BSA/BiNb was used as a

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photoelectrochemical immunosensor and incubated in NGAL (Ag, 25 µL) solution with a variety

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of concentrations at 37 ºC for 40 min. Finally, the electrode was rinsed with PBS, followed by the 6


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respective PEC measurement.

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Results and Discussion

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Bactrian Camel Immunization and Library Construction

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A healthy bactrian camel was immunized with NGAL toward the generation of

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affinity-matured anti-NGAL Nbs. As shown in Scheme 1, the blood of bactrian camel was

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collected after sixth immunization and used for RNA extraction. Then, the VHH sequences were

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amplified from the immunized bactrian camel’s lymphocyte cDNA. The VHH fragments were

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amplified with the ~ 600 bp fragment purified by gel extraction as the template (Figure 1A). Pst I

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and Not I were introduced to the 5’ and 3’ ends of the VHH fragments for library construction. In

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the library, the titer was 1.15×108 cfu mL-1, which is possible to obtain Nbs with high specificity

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and sequence diversity. To determine the percentage of library colonies containing correct

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insertion, PCR extension was performed on 24 individual colonies randomly selected from the

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library. The PCR screening shows that the correct insert ratio was nearly 100% (Figure 1B).

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Overall, we have successfully obtained an immune phage display library with high quality for the

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following selection of anti-NGAL Nbs.

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The anti-NGAL Nbs were identified by phage display technology from the constructed VHH

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library. Approximately 2×1011 phages were used in every round of panning. We calculated the

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relative enriching efficiency of phage particles eluted from wells coated with NGAL versus those

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without NGAL. The bar graph reveals the enrichment times and the ratios increase to 281-fold

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after the 3rd bio-panning (Figure 1C). A total of 95 individual colonies were randomly picked to

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identify NGAL-specific VHH by performing PE-ELISA. The positive colonies were sequenced,

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and these VHHs were classified into 4 families based on the diversity of amino acid sequences in

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CDR3 region (Figure 1D). We termed them Nb1, Nb2, Nb3 and Nb4.

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Nbs Expression, Purification and SPRi Binding Assay

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The display vectors pMECS permit the inducible periplasmic expression of Nbs as soluble

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C-terminally hexahistidine (His6)-tagged proteins in E.coli strain WK6. WK6 can suppress the

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amber stop codon between gene III and VHH gene on pMECS but TG1 cannot address this.

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Therefore, we transformed recombinant phagemid from TG1 to WK6 strain directly. Soluble Nbs

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were obtained with NI-NTA Superflow Sepharose columns. SDS-PAGE analysis demonstrates 7



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high quality of > 90% pure Nbs were obtained (Figure 1E). The yield of Nbs is shown in Figure

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2C with the ranging from 6.0 mg L-1 to 9.0 mg L-1. According to the result of SRPi binding assay,

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these 4 Nbs showed high affinity and strong binding to NGAL, and presented the affinities with

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equilibrium dissociation constant (KD) ranging from 8.16×10-8 to 2.09×10-9 (Figure. 2A, B).

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Thermostability of Nbs

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Thermostability of Nbs was performed by comparison to NGAL-specific Ab. A standard

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antigen selective ELISA was investigated to assess the retention of binding activity after various

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temperature for incubation. Antibody solutions of Nb1 and Ab were placed at 37 ºC for 0, 2, 12,

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24, 48, 72 and 96 hours. The activity of Nb1 and Ab before incubated at 37 ºC is regarded as

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100%. After treatment for 96 hours, the relative activity of Nb1 still keeps 92%. Ab gradually lost

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binding activity by increasing incubation time (Figure 2C). As shown in Figure 2D, the relative

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activity of both Nb1 and Ab decreases with increasing temperature and with increased duration at

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that temperature. Significantly, the Nb1 retains more than 90% of its binding activity when

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incubated at 95 ºC for 10 min and greater than 70% of its activity when incubated at 95 ºC for 60

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min, while the activity of Ab is below 10% after either 10 or 60 min of incubation at 95 ºC. These

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results are similar to studies that compared the thermostability of Nbs and conventional antibodies

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toward their respective antigens.34-36 These outstanding features of Nb would make it become

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promising diagnostic material for clinical application in harsh environments.

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Photoelectrochemical Properties of ITO/TiO2/CoPc Electrode

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A suitable system with enhanced photocurrent intensity and less electron-hole

21

recombination is critical for the application in photoelectrochemical sensing. The SEM

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morphologies of ITO/TiO2 electrode and ITO/TiO2/CoPc electrode are clearly displayed in Figure

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3A, B. It can be confirmed that the morphology of ITO/TiO2/CoPc electrode is similar to that of

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ITO/TiO2 electrode. In addition, the absorption of CoPc does not significantly affect the size of

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TiO2. The mean sizes of both nanoparticles are ca. 30 nm. The elemental composition (C, Ti, N,

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O, Co) of ITO/TiO2/CoPc electrode obtained from the energy dispersive spectrum (EDS) is

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presented in Figure 3C, which clearly indicates the presence of CoPc on the electrode surface.

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Upon irradiation with visible light, the ITO/TiO2 electrode shows a photocurrent of 2.44 µA

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cm-2 at an applied potential of -0.2 V (Figure 4A, curve a), whereas the ITO/TiO2/CoPc electrode 8


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shows a photocurrent of 10.7 µA cm-2 (Figure 4A, curve b), which indicates the improvement of

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the photo-current conversion efficiency of TiO2 by the absorption of CoPc because of the strong

3

electronic coupling between the excited-state of CoPc and the conduction band of TiO2. Ascorbic

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acid (AA), a nontoxic and efficient electron donor,26, 37 was further added to the electrolytes in

5

order to suppress the electron-hole recombination and therefore enhance the photocurrent

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intensity. The photocurrent of ITO/TiO2/CoPc electrode reaches up to 75.4 µA cm-2 (Figure 4A,

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curve c), which is ca. 7 times higher than that without AA (Figure 4A, curve b). The photocurrent

8

increases with increasing AA concentration up to 0.1 M, and then decreases (Supporting

9

Information, Figure S1). Therefore, a concentration of 0.1 M is employed for photocurrent

10

measurements.

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The photocurrent generation mechanism of the ITO/TiO2/CoPc electrode is illustrated in

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Figure 4B. The lowest unoccupied molecular orbital (LUMO) of CoPc is -3.9 eV,38 which lies

13

more positive than the conduction band (CB) level of TiO2 of -4.21 eV.39 Thus, the

14

photogenerated electrons from the LUMO of CoPc would quickly inject into the CB of TiO2, and

15

flow to the external circuit through the conductive ITO substrate under irradiation.

16

Simultaneously, holes in TiO2 valence band (VB) would transfer to the highest occupied

17

molecular orbital (HOMO) of CoPc, and are sacrificed by AA.38 Such a synergy effect in the

18

proposed ITO/TiO2/CoPc electrode could hasten the charge separation, suppress the electron-hole

19

recombination, and hence increase photocurrent intensity.

20

To optimize the thickness of TiO2 films on the surface of electrode, different concentrations

21

of TiO2 suspensions were employed to prepare ITO/TiO2/CoPc electrode and is shown in Figure

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S2. The photocurrent increases with increasing concentrations of TiO2 suspensions up to 2 mg

23

mL-1, and then a decreasing photocurrent is observed upon further increase of its concentration.

24

The thickness of TiO2 films could increase with increasing the concentration of TiO2 films,

25

leading to the increase of the surface area, which indicates that more amounts of CoPc would be

26

absorbed on thicker TiO2 films.40 Therefore, the photocurrent intensity increases with the

27

increase of TiO2 thickness because of the absorption of more amounts of CoPc. However, the

28

diffusion resistance for electron motion also increases in thicker due to the increase of the surface

29

recombination centers, resulting in a reduced photocurrent with thicker TiO2 films.40 Thus, the 9



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1

optimal concentration of 2 mg mL-1 TiO2 suspension was used to obtain ITO/TiO2/CoPc

2

electrode in following experiments. The immersion time of the ITO/TiO2 electrode in CoPc

3

solution is another important factor to affect the photoelectrochemical performance. The

4

photocurrent is enhanced with the increase of immersion time and trends to increase slowly after

5

4h (Figure S3). More absorption of CoPc in the ITO/TiO2 electrode enhances photocurrent

6

response. Therefore, 4h were employed as the optimum time for preparing the ITO/TiO2/CoPc

7

electrode.

8

Construction of Photoelectrochemical Immunosensor

9

In order to immobilize Nb to streptavidin-functionalized surfaces, Nb1, with the highest

10

affinity, was picked out to label with biotin in vivo. A label-free immunosensor was fabricated by

11

successive modifying the ITO/TiO2/CoPc electrode with CS, streptavidin (SA), BSA, and

12

biotinylated Nanobody (BiNb). The biosensor fabrication process was illustrated by the

13

electrochemical impedance spectroscopy (EIS), which is an effective way to monitor the

14

biosensor fabrication process.41 A bare ITO electrode shows the electron-transfer resistance (Ret)

15

value of 74 Ω (Figure 5A, curve a). With the successful assembly of TiO2, the Ret value decreases

16

to 93 Ω (Figure 5A, curve b). The Ret value of ITO/TiO2/CoPc electrode increases to 106 Ω due

17

to the absorption of CoPc on TiO2 films (Figure 5A, curve c). Subsequently, CS, SA, BSA, BiNb,

18

and NGAL (1 ng mL-1) were gradually immobilized on the electrode surface, and the value Ret of

19

each related electrode increase correspondingly to 121, 149, 182, 198 and 242 Ω (Figure 5A,

20

curve d, e, f, g), indicating the successful assembling of sensing on the electrode surface. The

21

reason for the Ret increase is that the nonconductive properties of the proteins obstruct the mass

22

transport and electron transfer of the redox probe to the electrode surface.41

23

The corresponding modification-induced changes in photocurrent are shown in Figure 5B.

24

Compare to the ITO electrode, the photocurrent increases significantly when the TiO2

25

nanoparticles was drop casted onto the ITO electrode (Figure 5B, curve a, b). It is found that the

26

photocurrent increases when the absorption of CoPc (Figure 5B, curve c), in which the generated

27

photocurrent is 3.4 times larger than ITO/TiO2 electrode. As expected, the successive binding of

28

CS, SA, BSA, and BiNb on the ITO/TiO2/CoPc electrode induces significant decreases in

29

photocurrent (Figure 5B, curve d, e, f, and g). After the as obtained immunosensor was incubated 10


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1

with NGAL (10 ng mL-1), the photocurrent intensity further decreases (Figure 5B, curve h). Such

2

decreases in photocurrent should be ascribed to the immobilized proteins induced steric

3

hindrances against the diffusion of AA to the photogenerated holes on the electrode interface.13

4

Control experiment, carrying out with immunosensor incubated in PBS solution without antigen,

5

shows no apparent change in photocurrent intensity (data not shown). These results thus

6

demonstrate that the resulting protein layer could greatly influence the photocurrent generation,

7

suggesting that a label-free photoelectrochemical immunosensor for quantitative detection of

8

NGAL is successfully achieved.

9

Detection of NGAL

10

Because the degree of signal decrement relates intimately with NGAL concentration, a

11

sensitive label-free NGAL biosensor can be achieved by tracking the final photocurrent variation

12

to monitor the immunoreaction extent of BiNb and NGAL. Figure 5C shows that the decrease in

13

photocurrent is dependent on the NGAL concentration. The relative change of photocurrent is

14

linearly proportional to the logarithm of the NGAL concentration in the range of 1 pg mL-1 to

15

500 ng mL-1 with a correlation coefficient of 0.9988, as shown in Figure 5D. The limit of

16

detection (LOD) is 0.6 pg mL-1 (S/N = 3). The sensitivity of the proposed photoelectrochemical

17

immunosensor here is much lower than those of previous methods such as enzyme-linked

18

immunosorbent assay (0.24 ng mL-1),5 and LSPR-based immunosensor (8.5 ng mL-1).8 The

19

improvement of the sensitivity could be ascribed to that the immobilization method for

20

immobilizing BiNb to streptavidin-functionalized surfaces to ensure a uniformed antibody

21

orientation with the antigen-binding sites toward the analytes, which resulted from higher binding

22

capacity to antigens.42-47

23

Specificity, reproducibility, stability and real sample analysis

24

The specificity of the proposed immunosensor was investigated by measuring the sensor

25

responses to HCG, CEA, PSA, and AFP at a concentration of 100 ng mL-1 that is 10-fold to

26

NGAL are shown in Figure 6. The photocurrent decrements from HCG, CEA, PSA, and AFP are

27

much lower than that of NGAL, indicating that the photocurrent reduction is mainly caused by a

28

specific antibody-antigen immunoreaction. The specificity of the proposed immunosensor was

29

also evaluated by measuring the photocurrent decrement of a mixed sample composed of 10 ng 11



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

1

mL-1 NGAL and 100 ng mL-1 HCG, CEA, PSA, and AFP, respectively. No significant change of

2

photocurrent difference could be observed as compared to the result obtained in the presence of

3

10 ng mL-1 NGAL only. The photocurrent is fairly reversible and very stable over 500 seconds

4

without any noticeable decrease, which suggests that the biosensor is suitable for PEC detection

5

(Figure S4). The results demonstrate that the proposed immunosensor has an acceptable

6

selectivity without apparent interference from nonspecific adsorption.

7

The reproducibility of an assay was expressed in terms of values for a within-batch

8

(intraassay) and a between-batch (interassay) relative standard deviation (RSD). A 10 ng mL-1

9

NGAL solution was repeatedly determined using the immunosensor for six times, giving an

10

intraassay RSD of 4.7%. The interassay RSD on six immunosensor is 5.9%. The results suggest a

11

good reproducibility of the proposed immunosensor.

12

Storage stability of the proposed immunosensor was investigated by storing the same patch

13

of ITO/TiO2/CoPc/CS/SA/BSA/BiNb electrode in 10 mM phosphate-buffered saline (PBS), pH

14

7.4, at 4 ºC under darkness for different periods before the PEC detection of 10 ng mL-1 NGAL. It

15

is clear that 93% of the initial signal for the immunosensor is retained after storage for four weeks,

16

indicating that the prepared immunosensor possesses good stability and potential for practical

17

application (Figure S5). The proposed immunosensor is applied to the detection of NGAL in

18

human serum samples (Table S1). The proposed method can detect NGAL in serum samples in a

19

wide concentration range of 0.01-100 ng mL-1, with good recoveries varied in the range of

20

93.2-112%, indicating this method has good sensitivity and reliability for detection of NGAL in

21

real serum samples.

22

Conclusions

23

In this work, we investigated a Nb-based PEC immunosensing strategy for sensitive NGAL

24

determination via specific recognition of NGAL on anti-NGAL Nbs orientedly modified

25

ITO/TiO2/CoPc electrode surface. An immunized NGAL Nb library was constructed, from which

26

four anti-NGAL Nbs with high affinity to NGAL have been obtained. Among them, Nb1 showing

27

the highest affinity was selected to conjugate with biotin successfully. The obtained Nb shows

28

better thermostability than the conventional antibody. The outstanding performance of Nb offers

29

attractive advantages in immunoassay development, not only allow a longer shelf life, but also 12


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1

allow effective and feasible test in harsh environment. With immobilization of BiNb on SA coated

2

CoPc-sensitized TiO2 electrode electrode surface, a label-free and highly sensitive PEC

3

immunosensor with a broad detection range and a low detection limit has been achieved, which

4

also reveals high specificity to NGAL. The CoPc-sensitized TiO2 electrode shows much higher

5

and stable photocurrent intensity that of pure TiO2 electrode since the CoPC accelerates the

6

migration of the photo-generated electrons. Immobilization of Nb provides high binding capacity,

7

and the LOD of the proposed method for NGAL detection is as low as 0.6 pg mL-1. The proposed

8

immunosensor with simple operation and rapid response from PEC would provide a promising

9

technique for ARF diagnosis with great clinical application potential.

10

Acknowledgment

11

This work was supported by Jiangsu Nanobody Engineering and Research Center of China

12

(Grant 2014-01), the National Basic Research Program of China (Grant 2010CB732400), the

13

National Natural Science Foundation of China (Grant 21035002, 21375014 and 21175021) and

14

the Fundamental Research Funds for the Central Universities (Grant 3207043903).

15

13



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Figure Captions

1 2

Scheme 1. Outline of strategies to select the anti-NGAL Nbs from an immunized bactrian camel

3

and to develop a label-free and sensitive immunosensor for NGAL based on anti-NGAL Nb using

4

the PEC technique.

5

Figure 1. Library construction and anti-NGAL Nbs selection. (A) The VHH genes were obtained

6

by two steps PCR and re-extracted by agarose gel purification. (B) 24 colonies were randomly

7

selected to estimate the correct insertion rate by performing PCR. (C) The enrichment factors of

8

phage particles eluted from wells coated with NGAL antigen versus those without antigen were

9

calculated after each round of panning. The bar graph presents the enriching ratio for every

10

panning. (D) Four classes of anti-NGAL VHHs containing different amino acid sequences

11

especially in CDR3 region were determined, named Nb1, Nb2, Nb3, and Nb4. Dots are used to

12

indicate residues identify to Nb1. (E) Four isolated Nbs were purified by NI-NTA Superflow

13

Sepharose column and analyzed by SDS-PAGE.

14

Figure 2. (A) KD between NGAL and four Nbs was determined by SPRi technique. NGAL

15

dilutions were injected at concentrations of 9.7, 29.2, 87.6 and 263 nM (C4 to C1). Each

16

sensorgram represents NGAL interacts with one class of immobilized anti-NGAL Nbs from the

17

lowest concentration (bottom curve, C4) to the highest concentration (top curve, C1). (B)

18

Relevant parameters of kinetic analysis. Thermal stability of the Nb1 and Ab. Antibodies were

19

incubated at 37 ºC for various lengths of time (0, 6, 12, 24, 48, 72, 96 h) (C) or at increasing

20

temperatures (25, 30, 37, 45, 75 and 95 ºC) for 10 min and 60 min (D), and activity was

21

determined by ELISA. Each test was assessed in triplicate.

22

Figure 3. SEM images of (A) ITO/TiO2 electrode and (B) ITO/TiO2/CoPc electrode. (C)

23

EDS spectrum of ITO/TiO2/CoPc electrode.

24

Figure 4. (A) Photocurrent response of (a) ITO/TiO2 electrode and (b, c) ITO/TiO2/CoPc

25

electrode in 0.1 M PBS (pH 7.4) in the (a, b) absence and (c) presence of 0.1 M AA at -0.2 V with

26

visible light excitation. (B) Schematic diagram of photocurrent generation mechanism of

27

ITO/TiO2/CoPc electrode.

28

Figure 5. (A) Nyquist diagrams of EIS, and (B) the corresponding photocurrent response of a

29

bare ITO electrode (a), the ITO/TiO2 electrode (b), the ITO/TiO2/CoPc electrode (c), the 17



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

electrode

(d),

the

ITO/TiO2/CoPc/CS/SA

Page 18 of 26

1

ITO/TiO2/CoPc/CS

2

ITO/TiO2/CoPc/CS/SA/BSA electrode (f), the ITO/TiO2/CoPc/CS/SA/BSA/BiNb electrode (g),

3

and the ITO/TiO2/CoPc/CS/SA/BSA/BiNb/NGAL (10 ng mL-1) electrode (h). (C) Effect of the

4

NGAL concentration on the differential photocurrent responses. I0 and I are the photocurrent of

5

the ITO/TiO2/CoPc/CS/SA/BSA/BiNb electrode before and after incubated with different

6

concentration of NGAL from 1 pg mL-1 to 500 ng mL-1. (D) The corresponding calibration curve

7

of the photcurrent decrement after incubation with the varied NGAL concentrations. The insert

8

regression equation is the linear relationship between the photocurrent decrement (I0-I) and the

9

logarithmic value of the NGAL concentration; R2 represents the correlation coefficient. The

10

photocurrent measurement was carried out in 0.1 M PBS (pH 7.4) containing 0.1 M AA at -0.2 V

11

with visible light excitation.

12

Figure 6. Selectivity of the proposed immunosensor to 10 ng mL-1 of NGAL by comparing it to

13

the interfering proteins at the 100 ng mL-1 level and the mixed sample. I0 and I are the

14

photocurrent of the ITO/TiO2/CoPc/CS/SA/BSA/BiNb electrode before and after incubated with

15

different proteins.

16

18


electrode

(e),

the

Page 19 of 26

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1 2

Scheme 1

3 4

19



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1 2

Figure 1

3 4

20


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1 2

Figure 2

3 4

21



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

1 2

Figure 3

3 4

22


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1 2

Figure 4

3 4

23



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1 2

Figure 5

3 4

24


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1 2

Figure 6

3

25



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For TOC only


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Label-Free Photoelectrochemical Immunosensor for Neutrophil

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