- Email: [email protected]
ELISA type fluorescence turn-on immunoassay based on Fe3+ induced fluorescence enhancement
Accepted Manuscript Title: ELISA type fluorescence turn-on immunoassay based on Fe3+ induced fluorescence enhancement Author: Zhongzhou Si Yining Li Hu Quan Haizhi Qi Ting Li Minghui Yang PII: DOI: Reference:
S0925-4005(14)00680-7 http://dx.doi.org/doi:10.1016/j.snb.2014.05.131 SNB 17005
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-1-2014 30-4-2014 29-5-2014
Please cite this article as: Z. Si, Y. Li, H. Quan, H. Qi, T. Li, M. Yang, ELISA type fluorescence turn-on immunoassay based on Fe3+ induced fluorescence enhancement, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.131 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ELISA type fluorescence turn-on immunoassay based on Fe3+ induced
2
fluorescence enhancement
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Zhongzhou Si a, 1, Yining Li a, 1, Hu Quan b, Haizhi Qi a, Ting Li a, *, Minghui Yang c,*
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These authors contributed equally to this work.
* Corresponding authors. Email: [email protected] (T. Li) [email protected] (M.Yang)
M
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University, Changsha, China 410011 b Department of Abdominal Surgery, The Affiliated Tumor Hospital of Xiang-ya School of Medicine, Central South University, Changsha, China 410000 c College of Chemistry and Chemical Engineering, Central South University, Changsha, China, 410083
d
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Department of General Surgery, The Second Xiang-ya Hospital, Central South
TeL: (+86) 731 88836356 (M.Yang)
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Abstract ELISA type fluorescence immunoassay for the sensitive detection of
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protein
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immuno-reaction was taken place in the wells of the 96 well plate using Fe3O4
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nanoparticles as label. The signal amplification strategy was based on the released
34
Fe3+ from Fe3O4 nanoparticles after acid treatment to trigger the fluorescence from
35
nonfluorescent fluorophore. The fluorophore selected was rhodamine 6G derivative
36
N-(rhodamine-6G)lactam-ethylenediamine (Rh6G-LEDA). Such signal turn-on
37
strategy has advantages of low background current and less susceptibility to
38
fluorescence quench. The immunoassay displays high sensitivity, wide linear range
39
and good selectivity for TNF-α detection. The developed immunoassay combines the
40
high throughput of ELISA type assay and the low cost as well as simplicity of
41
nanoparticle label, which could find wide applications for clinical screening purposes.
44 45 46 47
factor
α (TNF-α) was
proposed.
The
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necrosis
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tumor
Keywords: Fluorescence immunoassay; Fe3O4 nanoparticles; Tumor necrosis factor
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biomarker
α; Rhodamine; High throughput
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1. Introduction For sensitive detection of protein biomarkers in human fluidics, the most common
54
method is based on the sandwich type detection protocol that labeling the reporter
55
molecules with different tags and then measuring the signal of these tags[1-3] .
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Various techniques have been explored to detect the signals of these tags, such as
57
fluorescence, electrochemistry, quartz crystal microbalance (QCM), and so on[4-6].
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Among these techniques, fluorescence has the advantages of high sensitivity and high
59
throughput. A great number of tags with diverse spectral properties are available,
60
which include organic dyes, quantum dots, carbon dots and so on [7-9].
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Different strategies have been explored to enhance the signal of the fluorescence
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bioassay, for example, metal ion induced fluorescence enhancement. Zhong et al.
63
reported Cd2+ could trigger fluorescence from nonfluorescent metal-sensitive dyes of
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Fluo-4. When the metal ion binds with Fluo-4, it altered the structure of the
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fluorophore to obtain much higher quantum yields. Utilizing this phenomenon, they
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M
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achieved sensitive detection of Human IgG[10]. Metals ions have also been reported that
can
transform rhodamine-based
dyes from nonfluorescent to strong
fluorescent[11-13]. Based on this, Sun and co-workers reported the detection of Fe3+ using
rhodamine
6G
derivative,
N-(rhodamine-6G)lactam-ethylenediamine
70
(Rh6G-LEDA)[14]. Once bound to Fe3+, Rh6G-LEDA was converted into the
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open-cyclic form and resulted in strong fluorescent.
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In this work, utilizing the above mentioned reaction between Fe3+ and rhodamine
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6G derivative, we reported an ELISA-type fluorescence immunoassay for the
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detection of protein biomarker tumor necrosis factor α (TNF-α). Fe3O4 nanoparticles
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were used to label the detection anti-TNF-α antibodies (Ab2). Primary anti-TNF-α
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antibodies (Ab1) were immobilized into the wells of the 96 plate. After the sandwich
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immuno-reaction, acid was added into the well to dissolve the Fe3O4 to release Fe3+
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ions, which in returned could bind with Rh6G-LEDA and produce fluorescence. High
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sensitivity was obtained due to the significant amount of Fe3+ ions produced.
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Compared to conventional ELISA, no enzyme is involved in this bioassay. Hence, this
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method is cost-effective, simple and stable, which has potential to find wide
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applications in clinical testing.
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2. Experimental
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2.1. Apparatus and reagents
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Tumor necrosis factor α (TNF-α), goat anti-TNF-α antibodies and the ELISA kit
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for TNF-α were obtained from R&D Systems, Inc. (MN, USA). Fe3O4 magnetic beads
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(diameter of 500 nm) with carboxylic modification were from Allrun Nanoscience &
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Technology Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS, 10 mM) were
prepared by mixing NaH2PO4 and Na2HPO4. All other reagents were of analytical grade and deionized water was used throughout the study. A SpectraMax M5 plate reader (Molecular Devices, USA) and fluorescence
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spectrophotometer (Hitachi, F4600) were used for fluorescence measurement. The
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regular 96 well microtiter plates were used to develop the assay.
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2.2 Synthesis of Rh6G-LEDA
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The synthesis of the rhodamine derivative Rh6G-LEDA was according to the
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published method[15]. Typically, 500 mg of rhodamine 6G was dissolved in 10 mL of
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hot ethanol, followed by addition of 0.34 mL of ethylenediamine. The reaction
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mixture was refluxed for 6 hours till the fluorescence of the solution was disappeared.
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The solution was cooled to room temperature, and the precipitate was collected and
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2.3 Conjugating Ab2 onto Fe3O4
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washed with 10 mL of cold ethanol.
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To conjugate Ab2 onto Fe3O4, the Fe3O4 nanoparticles ( 1 mg/mL) were first
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mixed EDC and NHS ( 100 mM). The mixture was stirred for 4 h and centrifuged.
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The obtained Fe3O4 was then mixed with Ab2 solution (10 μg/mL ). After another 12 h
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of reaction, the solution was centrifuged again. The resulting Fe3O4-Ab2 bioconjugate
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was stored at 4 0C in phosphate buffer solution before use.
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2.4 Preparation of the immunoassay
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Typically, into the wells of the 96-well plate, 50 μL of Ab1 solution ( 10 μg/mL)
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was added and incubated at 4 °C overnight. After extensive wash with PBS, the wells
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were blocked with 1% BSA for 30 min. Then, 50 μL of different concentrations of TNF-α were added into the wells and incubated at 37 °C for 1 h. After another round of wash, 50 μL of the prepared Fe3O4-Ab2 bioconjugate was added into the wells and incubated at 37 °C for another 1 h. After wash again, the wells were ready for
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measurement.
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2.5 Detection protocol
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Into each well, 5 μL of HCl solution ( 1 mM) was added and incubated for 10 min.
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Then, 50 μL of the synthesized Rh6G-LEDA solution ( 10 μM dissolved in
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acetonitrile ) was added into the well. The fluorescence intensity of the wells was then
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measured.
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3. Results and discussion In conventional ELISA, enzymes, such as horseradish peroxidase (HRP) was used
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as catalytic label to generate the detection signal[16]. In this work, we replaced HRP
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with Fe3O4 nanoparticles for fluorescence immunoassay. Compared to HRP, Fe3O4
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nanoparticle has the advantages of low cost and better stability. Another advantage of
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using Fe3O4 nanoparticle as label in such fluorescence immunoassay is that there is no
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need to worry about fluorescence quenching of the fluorophores, as the fluorophores
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are present in the detection solution. In addition, they are barely fluorescent before
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bound with Fe3+. Fig. 1 shows the schematic representation of the immunoassay
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procedure.
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Figure 1
First, the reaction of Fe3+ with Rh6G-LEDA was investigated. As shown in Fig.
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2A, for Rh6G-LEDA solution (10 μM), almost no fluorescence emission was observed. While after the addition of 10 μM Fe3+, it can be seen the fluorescence emission exhibited about 15 times of enhancement. In addition, the fluorescence intensity was increased in proportional to the Fe3+ concentration (Fig. 2B), which
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indicated the possibility of applying this reaction for sensitive immunoassay. In our
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immunoassay design, the dissolution of the Fe3O4 label by HCl will release Fe3+,
138
which will trigger the fluorescence of Rh6G-LEDA.
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Figure 2
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The sandwich type immuno-reaction was taken step-by-step in the wells of the
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plate. Parameters that affect the sensitivity of the immunoassay were optimized, for
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example, the concentration of Rh6G-LEDA. For the detection of 1 ng/mL of TNF-α,
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it can be seen from Fig. 3A that increase the concentration of Rh6G-LEDA from 1
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μM to 5 μM, the sensitivity of the immunoassay increased and then reached a plateau.
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Further increase of the Rh6G-LEDA concentration will not improve the sensitivity of
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the immunoassay. On the contrary, the sensitivity was a little decreased. So the
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concentration of Rh6G-LEDA was chose as 5 μM. The definition of a
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signal-to-background (S/B) analysis is the signal of the sample (S) compared with that
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of the buffer without TNF-α (B). We think at this concentration, the released Fe3+ and
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Rh6G-LEDA reached an optimized ratio to obtain the highest fluorescence intensity.
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Under optimized experimental conditions, different concentrations of TNF-α were
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tested and the relationship between fluorescence intensity and TNF-α concentration
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were analyzed. The immunoassay displays wide linear range (0.01 to 10 ng/mL)
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towards TNF-α detection with sensitivity of about 1300 (a.u.)/ng.mL-1 (Fig. 3B). Based on signal to noise of 3, the detection limit of was found to be 5 pg/mL. The detection limit towards TNF-α is lower than surface plasmon resonance (SPR) immunoassay
based
on
gold
nanoparticles
(11.6
pg/mL)[17],
label-free
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electrochemical immunosensor based on K3[Fe(CN)6] as signal (10 pg/mL) [18] and
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electrochemical immunosensor using alkaline phosphatase (ALP) as signal label (10
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pg/mL)[19]. High sensitivity can be ascribed to the significant amount of Fe3+
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released when Fe3O4 nanoparticles were dissolved, and the strong fluorescence
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generated when Fe3+ were bound to Rh6G-LEDA. The selectivity of the immunoassay was investigated. The response of the
164
immunoassay to different potential interfering proteins, such as human IgG,
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carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and prostate specific
166
antigen (PSA) were studied. Solutions containing both TNF-α ( 1 ng/mL) and the
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above mentioned interfering proteins ( 10 ng/mL) were tested by the immunoassay.
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The response variations due to the interfering proteins are less than 10% of that to the
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1 ng/mL of TNF-α, indicating good selectivity of the immunoassay (Supporting
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Information, Fig. S1).
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To test the reproducibility of the immunoassay, 10 TNF-α samples ( 1 ng/mL)
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were analyzed simultaneously, and a relative standard deviation (RSD) of 4.3% was
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obtained. The good reproducibility was ascribed to the simplicity of the immunoassay
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process as well as the Fe3O4 based label.
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The stability of the Fe3O4 based label was also studied. When not in use, the
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label was stored in buffer at 4 0C. During one month, the same batch of label was used to develop immunoassays for TNF-α detection. In the first two weeks, no obvious signal variation was observed. After one month, around 90% of the initial response was still obtained (Supporting Information, Fig. S2), indicating good stability. The loss of signals can be ascribed to partially denaturing of the antibodies.
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Due to its good performance, the proposed immunoassay was applied for the
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analysis of serum samples (obtained from the Second Xiang-ya Hospital) and the
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results were compared with those determined by the standard ELISA kit. TNF-α
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concentrations determined by the two methods agreed well and the plot of TNF-α
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concentrations obtained by the two methods gave a straight line with a correlation
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coefficient of 0.993 (Figure 3C). The above data demonstrated good reliability of the
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immunoassay results, indicating potential application of the immunoassay for clinical
188
applications.
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4. Conclusions
In summary, by replacing the enzyme label in conventional ELISA assay with
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Fe3O4 nanoparticles, we developed a new fluorescence immunoassay for the detection
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of cancer biomarker TNF-α. Ab2 was directly conjugated onto Fe3O4 surface through
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covalent binding, without tedious process. Compared to labels based on enzymes or
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fluorescence fluorophores, the Fe3O4 nanoparticles have the advantages of low cost,
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simplicity and good stability. The proposed immunoassay can be easily adapted to the
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detection of other protein biomarkers.
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Acknowledgement
We are grateful for the support of National Natural Science Foundation of China
(21105128, 81200326).
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Biographies
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Zhongzhou Si, Ph.D, associate professor at the Second Xiang-ya Hospital, Central
275
South University. He obtained his Ph.D from the Second Xiang-ya Hospital, Central
276
South University at 2011. His research area is biomaterials and cancer biomarker
277
detection.
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Yining Li, B.S, he is a now a graduate student at the Second Xiang-ya Hospital,
280
Central South University. He obtained his B.S degree from the Hunan medical school
281
at 1996. His research interest includes biosensors and drug delivery.
M
an
279
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Hu Quan, Master, he is a surgeon at the Affiliated Tumor Hospital of Xiang-ya
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School of Medicine, Central South University. He obtained his Master degree from
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the Second Xiang-ya Hospital, Central South University at 2009. His research interest
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includes cancer biomarker detection.
Haizhi Qi, B.S, professor at the Second Xiang-ya Hospital, Central South University. He obtained his B.S from the Human medical school at 1983. His research includes biomaterials, biosensors and protein analysis.
291 292
Ting Li, Ph.D, lecturer at the Second Xiang-ya Hospital, Central South University.
293
He obtained his Ph.D from the Second Xiang-ya Hospital, Central South University at
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Page 13 of 18
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2011. His research interest includes cancer and transplantation biomarker detection
295
and protein analysis.
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Minghui Yang, Ph.D, professor at college of chemistry and chemical engineering, Central South University. He obtained his Ph.D from Hunan University at 2007. His research interest includes biosensors, biomaterials and microfluidics.
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Figure Captions:
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Figure 1. Schematic representation of the immunoassay procedure
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Figure 2. (A) Fluorescence emission of Rh6G-LEAD (10 μM, dissolved in
303
acetonitrile) in the absence (a) and presence of 10 μM of Fe3+. (B) Fluorescence
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emission of Rh6G-LEAD (10 μM) in the presence of different concentrations of
305
Fe3+. From a to h, 0, 2, 4, 6, 8, 10, 20, 30 μM. Excitation 500 nm.
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Figure 3. (A) Effect of Rh6G-LEAD concentration on the sensitivity of the
307
immunoassay towards the detection of 1 ng/mL of TNF-α. (B) Calibration
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curve of the immunoassay to different concentrations of TNF-α. (C) Compare
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the TNF-α concentration in serum determined by our immunoassay and by the
310
ELISA method. Error bar = RSD (n =3)
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HCl Fe3O4 TNF-α Ab1
Rh6G-LEDA =N-(rhodamine-6G)lactam-ethylenediamine
cr
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Rh6G-LEDA
Fe3+_Rh6G-LEDA
Fe3+
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Figure 1
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Ab2
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450
A
350 300
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b
150 100 50
a
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Fluorescence intensity (a.u.)
400
0 500 520 540 560 580 600 620 640 660 680
h
800 600 400
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B
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Fluorescence intensity (a.u.)
1000
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Wavelength (nm)
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200
a
0
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Wavelength (nm)
Figure 2
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500 520 540 560 580 600 620 640 660 680
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A
10 9 8
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S/B
7
5 4
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1
2 3 4 5 6 7 Rh6G-LEDA concentration, μM
8
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B
12 10 8
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S/B
cr
3
6
0.01
0.1
d
4 1
10
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TNF-α concentration, ng/mL
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Immunosensor results, pg/mL
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C
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25
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ELISA results, pg/mL
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Figure 3
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S0925-4005(14)00680-7 http://dx.doi.org/doi:10.1016/j.snb.2014.05.131 SNB 17005
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-1-2014 30-4-2014 29-5-2014
Please cite this article as: Z. Si, Y. Li, H. Quan, H. Qi, T. Li, M. Yang, ELISA type fluorescence turn-on immunoassay based on Fe3+ induced fluorescence enhancement, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.131 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ELISA type fluorescence turn-on immunoassay based on Fe3+ induced
2
fluorescence enhancement
3
ip t
Zhongzhou Si a, 1, Yining Li a, 1, Hu Quan b, Haizhi Qi a, Ting Li a, *, Minghui Yang c,*
4 5
20 21 22 23 24 25
cr
us
an
1
These authors contributed equally to this work.
* Corresponding authors. Email: [email protected] (T. Li) [email protected] (M.Yang)
M
13 14 15 16 17 18 19
University, Changsha, China 410011 b Department of Abdominal Surgery, The Affiliated Tumor Hospital of Xiang-ya School of Medicine, Central South University, Changsha, China 410000 c College of Chemistry and Chemical Engineering, Central South University, Changsha, China, 410083
d
12
Department of General Surgery, The Second Xiang-ya Hospital, Central South
TeL: (+86) 731 88836356 (M.Yang)
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a
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Page 1 of 18
30
Abstract ELISA type fluorescence immunoassay for the sensitive detection of
31
protein
32
immuno-reaction was taken place in the wells of the 96 well plate using Fe3O4
33
nanoparticles as label. The signal amplification strategy was based on the released
34
Fe3+ from Fe3O4 nanoparticles after acid treatment to trigger the fluorescence from
35
nonfluorescent fluorophore. The fluorophore selected was rhodamine 6G derivative
36
N-(rhodamine-6G)lactam-ethylenediamine (Rh6G-LEDA). Such signal turn-on
37
strategy has advantages of low background current and less susceptibility to
38
fluorescence quench. The immunoassay displays high sensitivity, wide linear range
39
and good selectivity for TNF-α detection. The developed immunoassay combines the
40
high throughput of ELISA type assay and the low cost as well as simplicity of
41
nanoparticle label, which could find wide applications for clinical screening purposes.
44 45 46 47
factor
α (TNF-α) was
proposed.
The
d
M
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necrosis
te
43
tumor
Keywords: Fluorescence immunoassay; Fe3O4 nanoparticles; Tumor necrosis factor
Ac ce p
42
biomarker
α; Rhodamine; High throughput
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Page 2 of 18
52
1. Introduction For sensitive detection of protein biomarkers in human fluidics, the most common
54
method is based on the sandwich type detection protocol that labeling the reporter
55
molecules with different tags and then measuring the signal of these tags[1-3] .
56
Various techniques have been explored to detect the signals of these tags, such as
57
fluorescence, electrochemistry, quartz crystal microbalance (QCM), and so on[4-6].
58
Among these techniques, fluorescence has the advantages of high sensitivity and high
59
throughput. A great number of tags with diverse spectral properties are available,
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which include organic dyes, quantum dots, carbon dots and so on [7-9].
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Different strategies have been explored to enhance the signal of the fluorescence
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bioassay, for example, metal ion induced fluorescence enhancement. Zhong et al.
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reported Cd2+ could trigger fluorescence from nonfluorescent metal-sensitive dyes of
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Fluo-4. When the metal ion binds with Fluo-4, it altered the structure of the
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fluorophore to obtain much higher quantum yields. Utilizing this phenomenon, they
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achieved sensitive detection of Human IgG[10]. Metals ions have also been reported that
can
transform rhodamine-based
dyes from nonfluorescent to strong
fluorescent[11-13]. Based on this, Sun and co-workers reported the detection of Fe3+ using
rhodamine
6G
derivative,
N-(rhodamine-6G)lactam-ethylenediamine
70
(Rh6G-LEDA)[14]. Once bound to Fe3+, Rh6G-LEDA was converted into the
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open-cyclic form and resulted in strong fluorescent.
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In this work, utilizing the above mentioned reaction between Fe3+ and rhodamine
73
6G derivative, we reported an ELISA-type fluorescence immunoassay for the
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detection of protein biomarker tumor necrosis factor α (TNF-α). Fe3O4 nanoparticles
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were used to label the detection anti-TNF-α antibodies (Ab2). Primary anti-TNF-α
76
antibodies (Ab1) were immobilized into the wells of the 96 plate. After the sandwich
77
immuno-reaction, acid was added into the well to dissolve the Fe3O4 to release Fe3+
78
ions, which in returned could bind with Rh6G-LEDA and produce fluorescence. High
79
sensitivity was obtained due to the significant amount of Fe3+ ions produced.
80
Compared to conventional ELISA, no enzyme is involved in this bioassay. Hence, this
81
method is cost-effective, simple and stable, which has potential to find wide
82
applications in clinical testing.
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2. Experimental
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2.1. Apparatus and reagents
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Tumor necrosis factor α (TNF-α), goat anti-TNF-α antibodies and the ELISA kit
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for TNF-α were obtained from R&D Systems, Inc. (MN, USA). Fe3O4 magnetic beads
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(diameter of 500 nm) with carboxylic modification were from Allrun Nanoscience &
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Technology Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS, 10 mM) were
prepared by mixing NaH2PO4 and Na2HPO4. All other reagents were of analytical grade and deionized water was used throughout the study. A SpectraMax M5 plate reader (Molecular Devices, USA) and fluorescence
92
spectrophotometer (Hitachi, F4600) were used for fluorescence measurement. The
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regular 96 well microtiter plates were used to develop the assay.
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2.2 Synthesis of Rh6G-LEDA
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The synthesis of the rhodamine derivative Rh6G-LEDA was according to the
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published method[15]. Typically, 500 mg of rhodamine 6G was dissolved in 10 mL of
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hot ethanol, followed by addition of 0.34 mL of ethylenediamine. The reaction
98
mixture was refluxed for 6 hours till the fluorescence of the solution was disappeared.
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The solution was cooled to room temperature, and the precipitate was collected and
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2.3 Conjugating Ab2 onto Fe3O4
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washed with 10 mL of cold ethanol.
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To conjugate Ab2 onto Fe3O4, the Fe3O4 nanoparticles ( 1 mg/mL) were first
103
mixed EDC and NHS ( 100 mM). The mixture was stirred for 4 h and centrifuged.
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The obtained Fe3O4 was then mixed with Ab2 solution (10 μg/mL ). After another 12 h
105
of reaction, the solution was centrifuged again. The resulting Fe3O4-Ab2 bioconjugate
106
was stored at 4 0C in phosphate buffer solution before use.
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2.4 Preparation of the immunoassay
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Typically, into the wells of the 96-well plate, 50 μL of Ab1 solution ( 10 μg/mL)
109
was added and incubated at 4 °C overnight. After extensive wash with PBS, the wells
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were blocked with 1% BSA for 30 min. Then, 50 μL of different concentrations of TNF-α were added into the wells and incubated at 37 °C for 1 h. After another round of wash, 50 μL of the prepared Fe3O4-Ab2 bioconjugate was added into the wells and incubated at 37 °C for another 1 h. After wash again, the wells were ready for
114
measurement.
115
2.5 Detection protocol
116
Into each well, 5 μL of HCl solution ( 1 mM) was added and incubated for 10 min.
117
Then, 50 μL of the synthesized Rh6G-LEDA solution ( 10 μM dissolved in
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118
acetonitrile ) was added into the well. The fluorescence intensity of the wells was then
119
measured.
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3. Results and discussion In conventional ELISA, enzymes, such as horseradish peroxidase (HRP) was used
122
as catalytic label to generate the detection signal[16]. In this work, we replaced HRP
123
with Fe3O4 nanoparticles for fluorescence immunoassay. Compared to HRP, Fe3O4
124
nanoparticle has the advantages of low cost and better stability. Another advantage of
125
using Fe3O4 nanoparticle as label in such fluorescence immunoassay is that there is no
126
need to worry about fluorescence quenching of the fluorophores, as the fluorophores
127
are present in the detection solution. In addition, they are barely fluorescent before
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bound with Fe3+. Fig. 1 shows the schematic representation of the immunoassay
129
procedure.
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Figure 1
First, the reaction of Fe3+ with Rh6G-LEDA was investigated. As shown in Fig.
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2A, for Rh6G-LEDA solution (10 μM), almost no fluorescence emission was observed. While after the addition of 10 μM Fe3+, it can be seen the fluorescence emission exhibited about 15 times of enhancement. In addition, the fluorescence intensity was increased in proportional to the Fe3+ concentration (Fig. 2B), which
136
indicated the possibility of applying this reaction for sensitive immunoassay. In our
137
immunoassay design, the dissolution of the Fe3O4 label by HCl will release Fe3+,
138
which will trigger the fluorescence of Rh6G-LEDA.
139
Figure 2
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The sandwich type immuno-reaction was taken step-by-step in the wells of the
141
plate. Parameters that affect the sensitivity of the immunoassay were optimized, for
142
example, the concentration of Rh6G-LEDA. For the detection of 1 ng/mL of TNF-α,
143
it can be seen from Fig. 3A that increase the concentration of Rh6G-LEDA from 1
144
μM to 5 μM, the sensitivity of the immunoassay increased and then reached a plateau.
145
Further increase of the Rh6G-LEDA concentration will not improve the sensitivity of
146
the immunoassay. On the contrary, the sensitivity was a little decreased. So the
147
concentration of Rh6G-LEDA was chose as 5 μM. The definition of a
148
signal-to-background (S/B) analysis is the signal of the sample (S) compared with that
149
of the buffer without TNF-α (B). We think at this concentration, the released Fe3+ and
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Rh6G-LEDA reached an optimized ratio to obtain the highest fluorescence intensity.
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Under optimized experimental conditions, different concentrations of TNF-α were
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tested and the relationship between fluorescence intensity and TNF-α concentration
153
were analyzed. The immunoassay displays wide linear range (0.01 to 10 ng/mL)
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towards TNF-α detection with sensitivity of about 1300 (a.u.)/ng.mL-1 (Fig. 3B). Based on signal to noise of 3, the detection limit of was found to be 5 pg/mL. The detection limit towards TNF-α is lower than surface plasmon resonance (SPR) immunoassay
based
on
gold
nanoparticles
(11.6
pg/mL)[17],
label-free
158
electrochemical immunosensor based on K3[Fe(CN)6] as signal (10 pg/mL) [18] and
159
electrochemical immunosensor using alkaline phosphatase (ALP) as signal label (10
160
pg/mL)[19]. High sensitivity can be ascribed to the significant amount of Fe3+
161
released when Fe3O4 nanoparticles were dissolved, and the strong fluorescence
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Page 7 of 18
162
generated when Fe3+ were bound to Rh6G-LEDA. The selectivity of the immunoassay was investigated. The response of the
164
immunoassay to different potential interfering proteins, such as human IgG,
165
carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and prostate specific
166
antigen (PSA) were studied. Solutions containing both TNF-α ( 1 ng/mL) and the
167
above mentioned interfering proteins ( 10 ng/mL) were tested by the immunoassay.
168
The response variations due to the interfering proteins are less than 10% of that to the
169
1 ng/mL of TNF-α, indicating good selectivity of the immunoassay (Supporting
170
Information, Fig. S1).
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To test the reproducibility of the immunoassay, 10 TNF-α samples ( 1 ng/mL)
172
were analyzed simultaneously, and a relative standard deviation (RSD) of 4.3% was
173
obtained. The good reproducibility was ascribed to the simplicity of the immunoassay
174
process as well as the Fe3O4 based label.
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The stability of the Fe3O4 based label was also studied. When not in use, the
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label was stored in buffer at 4 0C. During one month, the same batch of label was used to develop immunoassays for TNF-α detection. In the first two weeks, no obvious signal variation was observed. After one month, around 90% of the initial response was still obtained (Supporting Information, Fig. S2), indicating good stability. The loss of signals can be ascribed to partially denaturing of the antibodies.
181
Due to its good performance, the proposed immunoassay was applied for the
182
analysis of serum samples (obtained from the Second Xiang-ya Hospital) and the
183
results were compared with those determined by the standard ELISA kit. TNF-α
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Page 8 of 18
concentrations determined by the two methods agreed well and the plot of TNF-α
185
concentrations obtained by the two methods gave a straight line with a correlation
186
coefficient of 0.993 (Figure 3C). The above data demonstrated good reliability of the
187
immunoassay results, indicating potential application of the immunoassay for clinical
188
applications.
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Figure 3
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4. Conclusions
In summary, by replacing the enzyme label in conventional ELISA assay with
192
Fe3O4 nanoparticles, we developed a new fluorescence immunoassay for the detection
193
of cancer biomarker TNF-α. Ab2 was directly conjugated onto Fe3O4 surface through
194
covalent binding, without tedious process. Compared to labels based on enzymes or
195
fluorescence fluorophores, the Fe3O4 nanoparticles have the advantages of low cost,
196
simplicity and good stability. The proposed immunoassay can be easily adapted to the
197
detection of other protein biomarkers.
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Acknowledgement
We are grateful for the support of National Natural Science Foundation of China
(21105128, 81200326).
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Biographies
274
Zhongzhou Si, Ph.D, associate professor at the Second Xiang-ya Hospital, Central
275
South University. He obtained his Ph.D from the Second Xiang-ya Hospital, Central
276
South University at 2011. His research area is biomaterials and cancer biomarker
277
detection.
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Yining Li, B.S, he is a now a graduate student at the Second Xiang-ya Hospital,
280
Central South University. He obtained his B.S degree from the Hunan medical school
281
at 1996. His research interest includes biosensors and drug delivery.
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Hu Quan, Master, he is a surgeon at the Affiliated Tumor Hospital of Xiang-ya
284
School of Medicine, Central South University. He obtained his Master degree from
285
the Second Xiang-ya Hospital, Central South University at 2009. His research interest
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includes cancer biomarker detection.
Haizhi Qi, B.S, professor at the Second Xiang-ya Hospital, Central South University. He obtained his B.S from the Human medical school at 1983. His research includes biomaterials, biosensors and protein analysis.
291 292
Ting Li, Ph.D, lecturer at the Second Xiang-ya Hospital, Central South University.
293
He obtained his Ph.D from the Second Xiang-ya Hospital, Central South University at
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Page 13 of 18
294
2011. His research interest includes cancer and transplantation biomarker detection
295
and protein analysis.
296
Minghui Yang, Ph.D, professor at college of chemistry and chemical engineering, Central South University. He obtained his Ph.D from Hunan University at 2007. His research interest includes biosensors, biomaterials and microfluidics.
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Figure Captions:
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Figure 1. Schematic representation of the immunoassay procedure
302
Figure 2. (A) Fluorescence emission of Rh6G-LEAD (10 μM, dissolved in
303
acetonitrile) in the absence (a) and presence of 10 μM of Fe3+. (B) Fluorescence
304
emission of Rh6G-LEAD (10 μM) in the presence of different concentrations of
305
Fe3+. From a to h, 0, 2, 4, 6, 8, 10, 20, 30 μM. Excitation 500 nm.
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Figure 3. (A) Effect of Rh6G-LEAD concentration on the sensitivity of the
307
immunoassay towards the detection of 1 ng/mL of TNF-α. (B) Calibration
308
curve of the immunoassay to different concentrations of TNF-α. (C) Compare
309
the TNF-α concentration in serum determined by our immunoassay and by the
310
ELISA method. Error bar = RSD (n =3)
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HCl Fe3O4 TNF-α Ab1
Rh6G-LEDA =N-(rhodamine-6G)lactam-ethylenediamine
cr
321
Rh6G-LEDA
Fe3+_Rh6G-LEDA
Fe3+
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Figure 1
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Ab2
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339
450
A
350 300
200
ip t
250
b
150 100 50
a
cr
Fluorescence intensity (a.u.)
400
0 500 520 540 560 580 600 620 640 660 680
h
800 600 400
an
B
M
Fluorescence intensity (a.u.)
1000
us
Wavelength (nm)
340
200
a
0
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Wavelength (nm)
Figure 2
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d
500 520 540 560 580 600 620 640 660 680
348 349
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Page 17 of 18
A
10 9 8
6
ip t
S/B
7
5 4
0
1
2 3 4 5 6 7 Rh6G-LEDA concentration, μM
8
9
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350
14
an
B
12 10 8
M
S/B
cr
3
6
0.01
0.1
d
4 1
10
te
TNF-α concentration, ng/mL
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Immunosensor results, pg/mL
40
C
35 30 25 20 15
15
20
25
30
35
ELISA results, pg/mL
352 353
Figure 3
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Page 18 of 18