Comparison of protein immobilization methods with covalent bonding on paper for paper-based enzyme-linked immunosorbent assay

Prévia do material em texto

Vol.:(0123456789)
Analytical and Bioanalytical Chemistry 
https://doi.org/10.1007/s00216-024-05575-4
RESEARCH PAPER
Comparison of protein immobilization methods with covalent 
bonding on paper for paper‑based enzyme‑linked immunosorbent 
assay
Yang Chen1 · Kaewta Danchana1  · Takashi Kaneta1 
Received: 30 June 2024 / Revised: 23 September 2024 / Accepted: 30 September 2024 
© The Author(s) 2024
Abstract
In this study, two methods were examined to optimize the immobilization of antibodies on paper when conducting a paper-
based enzyme-linked immunosorbent assay (P-ELISA). Human IgG, as a test-capture protein, was immobilized on paper 
via the formation of Schiff bases. Aldehyde groups were introduced onto the surface of the paper via two methods: NaIO4 
and 3-aminopropyltriethoxysilane (APTS) with glutaraldehyde (APTS-glutaraldehyde). In the assay, horseradish peroxi-
dase-conjugated anti-human IgG (HRP-anti-IgG) binds to the immobilized human IgG, and the colorimetric reaction of 
3,3′,5,5′-tetramethylbenzyzine (TMB) produces a blue color in the presence of H2O2 and HRP-anti-IgG as a model analyte. 
The immobilization of human IgG, the enzymatic reaction conditions, and the reduction of the chemical bond between the 
paper surface and immobilized human IgG all were optimized in order to improve both the analytical performance and the 
stability. In addition, the thickness of the paper was examined to stabilize the analytical signal. Consequently, the APTS-
glutaraldehyde method was superior to the NaIO4 method in terms of sensitivity and reproducibility. Conversely, the reduc-
tion of imine to amine with NaBH4 proved to exert only minimal influence on sensitivity and stability, although it tended to 
degrade reproducibility. We also found that thick paper was preferential when using P-ELISA because a rigid paper substrate 
prevents distortion of the paper surface that is often caused by repeated washing processes.
Keywords Paper-based enzyme-linked immunosorbent assay · ELISA · Immobilization · Covalent bonding · Protein
Introduction
Enzyme-linked immunosorbent assay (ELISA) has become 
a popular method of detection because it is highly selec-
tive, or specific, to target analytes and it lends a more high-
throughput nature to the process [1, 2]. The method is based 
on an immunological reaction where immobilized antibodies 
bind to the target analyte. This is followed by an enzymatic 
reaction where the enzyme catalyzes the conversion of a 
substrate to a product, which thereby enhances the detec-
tion sensitivity. Therefore, ELISA is generally specific and 
sensitive to the target analyte [3].
There are many types of ELISA that generally include 
homogeneous, competitive, and sandwich assays [4]. In 
these methods, different enzymes are conjugated with the 
antibodies of analytes, such as lysozyme, malate dehydro-
genase, or glucose-6-phosphate dehydrogenase. A suit-
able substrate is selected to enhance the sensitivity of the 
method, and that is dependent on the enzyme that is linked 
with either the antigen or the antibody. One of the most fre-
quently employed enzyme-substrate combinations is horse-
radish peroxidase and 3,3′,5,5′-tetramethylbenzidine [5].
Commercially available ELISA kits are based on sand-
wich assays that utilize a microtiter plate. The sandwich 
assay involves binding a target analyte to an immobilized 
antibody on the walls of each well, which is followed by a 
probe antibody binding the analyte at a different site from 
that of the immobilized antibody. If the probe antibody is not 
linked with the enzyme, a secondary antibody linked with 
the enzyme recognizes it. As a result, the enzymatic reaction 
with a substrate determines the amount of probe antibody 
corresponding to the analyte. A microplate reader is used 
to measure the absorbance, fluorescence, or chemilumines-
cence, depending on the properties of the enzymatic product.
 * Takashi Kaneta 
 kaneta@okayama-u.ac.jp
1 Department of Chemistry, Okayama University, 3-1-1 
Tsushimanaka, Kita-ku, Okayama 700-8530, Japan
 Y. Chen et al.
However, microplate readers are expensive instruments, 
and commercially available ELISA kits consume expensive 
microtiter plates and large volumes of substrate solution. 
Therefore, only well-equipped laboratories are able to use 
commercially available ELISA kits. Conversely, the use of a 
paper-based ELISA (P-ELISA) reduces the cost of tools and 
detection instruments [6]. For example, Verma et al. estimate 
the price of a P-ELISA at $2.15 [7], which is much cheaper 
than a commercially available ELISA kit; an ELISA kit for 
albumin is roughly $1,000 for 98 wells, which amounts to 
$10 for one sample. Conversely, P-ELISA necessitates man-
ual data processing, which could lead to errors greater than 
those of microplate readers. However, P-ELISA is useful for 
determining a rough range for the concentration of a target 
analyte. Thus, P-ELISA has attracted much attention as a 
platform that could be an alternative to the use of microtiter 
plates.
The P-ELISA was first reported by Chen et al. in 2010. 
Those researchers used photolithography and used a hydro-
phobic barrier to restrict the detection zone [6]. They immo-
bilized a target protein in the detection zone via nonspecific 
adsorption and detected it via the use of either fluorescently 
labeled or enzyme-labeled antibodies that permits the meas-
urement of a target protein. Many studies use the physical 
adsorption of proteins [4–15] and bacteria [16] on paper sub-
strates. Paper substrates are frequently coated with chitosan 
to introduce amino groups on the paper surface [17–21]. 
Amino groups of chitosan are coupled with glutaraldehyde 
to immobilize capture proteins.
Conversely, immobilization of proteins by covalent bond-
ing is attractive since it is more stable than the physical 
adsorption against the washing process when using ELISA, 
and stability also is a factor in the long-term storage of 
devices. The immobilization of capture proteins on paper 
requires modification of the functional groups to produce 
groups of aldehydes, aminos, or carboxyls. The antibody 
to a target molecule is immobilized covalently using alde-
hyde groups that are produced via the oxidation of hydroxyl 
groups to aldehyde groups with sodium periodate [22, 23]. 
A silane coupling reagent, 3-aminopropyltriethoxysilane 
(APTS), is also a conventional reagent that is used to intro-
duce amino groups onto paper [24, 25]. Paper substrates are 
treated with APTS, followed by a reaction with glutaralde-
hyde to introduce aldehyde groups onto paper. The aldehyde 
groups react with the amino groups of proteins to immobi-
lize them onto paper.
Obviously, the immobilization efficiency of capture pro-
teins is one of the key issues that determines the performance 
of P-ELISA because the sensitivity would be directly influ-
enced by the number of proteins that ultimately is retained 
on the paper. Therefore, in this study, we investigated the 
immobilization of proteins using APTS and glutaraldehyde 
and compared the results with a method that uses sodium 
periodate. We used horseradish peroxidase-labeled anti-
human IgG (HRP-anti-IgG) as a model analyte to compare 
the sensitivity and stability of these immobilization meth-
ods. Although the method cannot be directly applied to real 
samples because HRP-anti-IgG is not a natural substance, 
this process permits the assessment of binding onto human 
IgG immobilized on paper. Human IgG antibody was immo-
bilized on a paper surface modified by APTS with glutaral-
dehyde and sodium periodate, and the immobilized human 
IgG was bound to HRP-anti-IgG, which was detected via 
the enzymatic reaction of HRP. The conditions for the enzy-
matic reaction and the thickness of the paper substrates were 
also optimized to obtain the best performance.
Materials and methods
Materials
Whatman cellulose papers (Grade 1 CHR sheets (thickness, 
0.1 mm; pore size,3MM CHR 
sheets (thickness, 0.34 mm; pore size,min when adding 4 μL 
of the TMB solution. Conversely, the addition of 7 μL rep-
resents the maximum value at 10 min, and the color inten-
sity began to decrease after 10 min. When the TMB volume 
was 10 μL, the color intensity reached its maximum and 
remained constant.
The decrease in color intensity is attributed to the disap-
pearance of the color with the evaporation of water. The 
oxidized TMB, which is the colored product, bleaches when 
being completely dried. Therefore, in volumes of 4 and 7 
μL, water began to evaporate at 4 and 10 min, respectively, 
which resulted in the initiation of color disappearance. 
Therefore, 10 μL of TMB maintained a stable intensity even 
after 10 min because this was the volume that was sufficient 
to moisturize the test zone.
Comparison of immobilization methods
The amount of immobilized protein directly influences the 
sensitivity of P-ELISA. Thus, in the present study, immobi-
lization methods were compared using the reaction between 
human IgG and HRP-anti-IgG. Physical adsorption is some-
times employed for P-ELISA because it has the advantages 
of being easy to operate and reducing the cost. However, 
immobilization via covalent bonding is more stable than 
physical adsorption, as demonstrated by Zhu and co-workers 
[22]. Therefore, covalent bonding would be more advanta-
geous in terms of protein immobilization yield, sensitivity, 
and stability. Thus, two types of modifications were exam-
ined in this study, one with NaIO4 and the other with APTS 
and glutaraldehyde, to produce aldehyde groups that are 
covalently bound with the amino groups of proteins.
In general, a calibration curve of P-ELISA is the rela-
tionship between the logarithm of the analyte concentra-
tion and the color intensity, and this results in a sigmoidal 
curve [6, 30]. However, to assess the binding ability of the 
immobilized human IgG, binding curves were constructed 
in this study by directly plotting the color intensity against 
the concentration of HRP-anti-IgG. The dependence of 
color intensity on the concentration of HRP-anti-IgG is 
shown in Fig. 3 where the concentration of HRP-anti-IgG 
was varied within a range of 0.5 to 2.0 μg mL−1. Figure 3a 
and b show the relationship between the concentration of 
HRP-anti-IgG and the color intensity obtained via immo-
bilization methods using NaIO4 and APTS-glutaraldehyde, 
respectively. In both cases, the color intensity increased 
with increases in the concentration of HRP-anti-IgG from 
0.5 to 1.0 μg mL−1. The intensity when using NaIO4, how-
ever, seemed to be saturated within a range of concentra-
tions of more than 0.5 μg mL−1. When comparing the two 
methods, the APTS-glutaraldehyde method more clearly 
showed the dependence of the color intensities on the con-
centration of HRP-anti-IgG. In addition, the correlation 
coefficient and sensitivity also were larger when using the 
APTS-glutaraldehyde method compared with the NaIO4 
method. The slopes, which indicate the sensitivity, can be 
directly related to the amount of the analyte bound to the 
immobilized antibodies; therefore, a large slope is prefer-
ential to sensitive detection. The limits of detection were 
estimated to be 0.65 μg mL−1 for the NaIO4 method and 
0.42 μg mL−1 for the APTS-glutaraldehyde method. These 
results imply that the immobilized efficiency of human IgG 
is greater when using the APTS-glutaraldehyde method. 
Therefore, the APTS-glutaraldehyde method is preferential 
to the NaIO4 method for P-ELISA in order to obtain the 
best analytical performance.
Fig. 3 Comparison of two immobilization methods for HRP-anti- IgG 
assay. a Oxidization with NaIO4 and b APTS and glutaraldehyde. The 
concentrations of HRP-anti-IgG were 0.4, 0.5, 0.75, 1.0, and 2.0 μg 
 mL−1. The error bars indicate the standard deviations for three repli-
cated measurements
 Y. Chen et al.
Effect of reduction with  NaBH4
The Schiff's base reaction generates a relatively unstable 
imine that must be stabilized by the reduction of imine to 
amine. In fact, reports in the literature depict how NaBH4 
reduces imines to amines and stabilizes the immobilized 
proteins [27, 31]. The effect of reduction was evaluated 
by comparing the binding curves for the P-ELISA devices 
with and without treatment with NaBH4. The binding curves 
showed no significant difference in a linear range of 0 to 1 
μg mL−1, whereas the slope and the correlation coefficient 
were slightly larger with NaBH4 (35.9 and R2 = 0.9816) than 
that without NaBH4 (32.6 and R2 = 0.959) (Supplementary 
material, Fig. S2). Conversely, the standard deviation of each 
data point was larger with reduction than without, which 
could be attributed to poor reproducibility in the reduction 
when using paper. Therefore, these results suggest that the 
reduction treatment somewhat degrades the reproducibility 
of P-ELISA because the sensitivity and linear range show a 
less significant difference.
The effect of reduction on the stability of the P-ELISA 
device was also evaluated via immunoassays using 1 μg 
 mL−1 HRP-anti-IgG. The P-ELISA devices were prepared 
on the same day and stored at 4 °C in a refrigerator. The 
results are shown in Fig. 4. It was apparent that the color 
intensity seemed to remain unchanged within 15 days for 
both devices, with and without reduction, and began to 
decrease after 16 days. We have also confirmed that the color 
intensity remained stable for only 3 days when the devices 
were stored at room temperature.
We also used a t-test to assess the stability of the device 
and compared the results of each day with those of the first 
day. The t-test results were slightly different from those 
obtained via visual assessment, as shown in Fig. 4. The 
 NaBH4 treatment showed no significant difference from the 
first day to the eighth day, but the device appeared to gradu-
ally deteriorate from the 11th day. On the other hand, with-
out the NaBH4 treatment, there was a significant difference 
from the first day to the second and third days, but no sig-
nificant difference from the fourth to the fifteenth day. Since 
the results from the fourth to the fifteenth day did not show a 
significant difference in the untreated device, it is likely that 
the reduction with NaBH4 would have less of an impact on 
stability. Conversely, the significant difference observed in 
the second and third days should be attributed to the small 
sample size of the t-test (it should normally be larger than 
30), the colorimetric reagents, or the experimental operation. 
Therefore, the reduction of the imine bond to an amine bond 
between the paper surface and human IgG had no significant 
influence on the stability of the P-ELISA device.
Effect of paper thickness
Two types of paper substrates were compared using the 
APTS-glutaraldehyde method. Although Grade 1 CHR 
and Grade 3MM CHR paper represent the same sensitiv-
ity (Fig. 3b and Fig. 5), the error bars of the 3MM CHR 
paper were significantly smaller than those of the Grade 1 
CHR. The LOD was also improved to 0.11 μg mL−1 when 
using Grade 3MM CHR paper. The rigidity of the paper 
substrate contributes to the good reproducibility of Grade 
Fig. 4 Effect of reduction with NaBH4 on the stability of ELISA. The 
concentration of HRP-anti-IgG, 1.0 μg mL−1
Fig. 5 Images of the P-ELISA devices and binding curve for HRP-
anti-IgG. Linear range, 0.05–2.0 μg mL−1
Comparison of protein immobilization methods with covalent bonding on paper for paper‑based…
3MM CHR paper. The thin paper substrate, Grade 1 CHR, 
distorted the surface when it was repeatedly washed with 
solutions, which resulted in poor reproducibility of the 
color intensity. However, the thick paper substrate, Grade 
3MM CHR, maintained a flat surface after being washed 
repeatedly. In addition, the linear range was expanded 
to 2 μg mL−1 when using the thick paper. Therefore, the 
thicker paper is preferential when using P-ELISA devices 
that must be washed many times.Conclusions
In this work, we compared two immobilization methods 
using NaIO4 and APTS-glutaraldehyde to produce alde-
hyde groups on paper substrates. We established that the 
APTS-glutaraldehyde method is superior to the NaIO4 
method in terms of reproducibility and sensitivity. We also 
found that a reduction with NaBH4 showed no difference in 
stability and instead degraded reproducibility, although it 
exhibited a slight improvement in sensitivity. The devices 
could be stored for 15 days at 4 °C in a refrigerator with-
out the need to reduce the imino bond to an amino bond. 
Good linearity was obtained within a range of 0 to 1.0 μg 
 mL−1 of HRP-anti-IgG. The effect of paper thickness was 
also examined, and a thicker paper substrate proved to be 
preferential when using P-ELISA due to the rigidity that 
prevents bending caused by the washing process. The pre-
sent work should provide useful information to help obtain 
the best performance when immobilizing antibodies on 
paper for P-ELISA. The developed device cannot directly 
be applied to real samples because the analyte used in this 
study is not found in real samples. However, this immo-
bilization method is expandable to sandwich assays when 
performing P-ELISA for an analyte in real samples.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00216- 024- 05575-4.
Author contributions Conceptualization: Takashi Kaneta. Data cura-
tion: Yang Chen. Formal analysis: Yang Chen, Kaewta Danchana, 
Takashi Kaneta. Funding acquisition: Kaewta Danchana, Takashi 
Kaneta. Investigation: Yang Chen, Kaewta Danchana, Takashi Kan-
eta. Methodology: Yang Chen, Takashi Kaneta. Project administration: 
Takashi Kaneta. Resources: Takashi Kaneta. Supervision: Kaewta Dan-
chana, Takashi Kaneta. Validation: Yang Chen. Visualization: Yang 
Chen, Takashi Kaneta. Writing — original draft: Yang Chen. Writing 
— review and editing: Takashi Kaneta
Funding Open Access funding provided by Okayama University. This 
work was supported by the Kurita Water and Environment Foundation 
(KWEF) Grant Number 23H035.
Declarations 
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long 
as you give appropriate credit to the original author(s) and the source, 
provide a link to the Creative Commons licence, and indicate if changes 
were made. The images or other third party material in this article are 
included in the article’s Creative Commons licence, unless indicated 
otherwise in a credit line to the material. If material is not included in 
the article’s Creative Commons licence and your intended use is not 
permitted by statutory regulation or exceeds the permitted use, you will 
need to obtain permission directly from the copyright holder. To view a 
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
 1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay 
(ELISA) Quantitative assay of immunoglobulin G. Immu-
nochemistry. 1971;8:871–4. https:// doi. org/ 10. 1016/ 0019- 
2791(71) 90454-x.
 2. Engvall E, Jonsson K, Perlmann P. Enzyme-linked immunosorb-
ent assay. II. Quantitative assay of protein antigen, immuno-
globulin G, by means of enzyme-labelled antigen and antibody-
coated tubes. Biochim Biophys Acta. 1971;251:427–34. https:// 
doi. org/ 10. 1016/ 0005- 2795(71) 90132-2.
 3. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays 
with special reference to ELISA techniques. J Clin Pathol. 
1978;31:507–20. https:// doi. org/ 10. 1136/ jcp. 31.6. 507.
 4. Aydin S. A short history, principles, and types of ELISA, and 
our laboratory experience with peptide/protein analyses using 
ELISA. Peptides. 2015;72:4–15. https:// doi. org/ 10. 1016/j. pepti 
des. 2015. 04. 012.
 5. Barry TP, Lapp AF, Kayes TB, Malison JA. Validation of a 
microtitre plate ELISA for measuring cortisol in fish and 
comparison of stress responses of rainbow trout (Oncorhyn-
chus mykiss) and lake trout (Salvelinus namaycush). Aquac. 
1993;117:351–63. https:// doi. org/ 10. 1016/ 0044- 8486(93) 
90331-R.
 6. Cheng CM, Martinez AW, Gong J, Mace CR, Phillips ST, 
Carrilho E, Mirica KA, Whitesides GM. Paper-based ELISA. 
Angew Chem Int Ed Engl. 2010;49:4771–4. https:// doi. org/ 10. 
1002/ anie. 20100 1005.
 7. Verma MS, Tsaloglou M-N, Sisley T, Christodouleas D, Chen 
A, Milettea J, Whitesides GM. Sliding-strip microfluidic device 
enables ELISA on paper. Biosens Bioelectron. 2018;99:77–84. 
https:// doi. org/ 10. 1016/j. bios. 2017. 07. 034.
 8. Murdock RC, Shen L, Griffin DK, Kelley-Loughnane N, 
Papautsky I, Hagen JA. Optimization of a paper-based 
ELISA for a human performance biomarker. Anal Chem. 
2013;85(23):11634–42. https:// doi. org/ 10. 1021/ ac403 040a.
 9. Hsu CK, Huang HY, Chen WR, Nishie W, Ujiie H, Natsuga K, 
Fan ST, Wang HK, Lee JY, Tsai WL, Shimizu H, Cheng CM. 
Paper-based ELISA for the detection of autoimmune antibod-
ies in body fluid-the case of bullous pemphigoid. Anal Chem. 
2014;86:4605–10. https:// doi. org/ 10. 1021/ ac500 835k.
 10. Khan MS, Pande T, van de Ven TG. Qualitative and quantita-
tive detection of T7 bacteriophages using paper based sand-
wich ELISA. Colloids Surf B Biointerfaces. 2015;132:264–70. 
https:// doi. org/ 10. 1016/j. colsu rfb. 2015. 05. 028.
 11. Hsu MY, Hung YC, Hwang DK, Lin SC, Lin KH, Wang CY, 
Choi HY, Wang YP, Cheng CM. Detection of aqueous VEGF 
concentrations before and after intravitreal injection of anti-
VEGF antibody using low-volume sampling paper-based 
ELISA. Sci Rep. 2016;6:34631. https:// doi. org/ 10. 1038/ srep3 
4631.
 Y. Chen et al.
 12. Wu D, Zhang J, Xu F, Wen X, Li P, Zhang X, Qiao S, Ge S, 
Xia N, Qian S, Qiu X. A paper-based microfluidic Dot-ELISA 
system with smartphone for the detection of influenza A. 
Microfluid Nanofluid. 2017;21:43. https:// doi. org/ 10. 1007/ 
s10404- 017- 1879-6.
 13. Kasetsirikul S, Umer M, Soda N, Sreejith KR, Shiddiky MJA, 
Nguyen NT. Detection of the SARS-CoV-2 humanized antibody 
with paper-based ELISA. Analyst. 2020;145:7680–6. https:// 
doi. org/ 10. 1039/ d0an0 1609h.
 14. Zhao Y, Zeng D, Yan C, Chen W, Ren J, Jiang Y, Jiang L, 
Xue F, Ji D, Tang F, Zhou M, Dai J. Rapid and accurate detec-
tion of Escherichia coli O157:H7 in beef using microfluidic 
wax-printed paper-based ELISA. Analyst. 2020;145:3106–15. 
https:// doi. org/ 10. 1039/ d0an0 0224k.
 15. Lee D, Asmare N, Sarioglu AF. Paper-based multi-well depletion 
ELISA. Lab Chip. 2023;23:251–60. https:// doi. org/ 10. 1039/ d2lc0 
0960a.
 16. Shih CM, Chang CL, Hsu MY, Lin JY, Kuan CM, Wang HK, 
Huang CT, Chung MC, Huang KC, Hsu CE, Wang CY, Shen YC, 
Cheng CM. Paper-based ELISA to rapidly detect Escherichia coli. 
Talanta. 2015;145:2–5. https:// doi. org/ 10. 1016/j. talan ta. 2015. 07. 
051.
 17. Wang S, Ge L, Song X, Yu J, Ge S, Huang J, Zeng F. Paper-based 
chemiluminescence ELISA: lab-on-paper based on chitosan modi-
fied paper device and wax-screen-printing. Biosens Bioelectron. 
2012;31:212–8. https:// doi. org/ 10. 1016/j. bios. 2011. 10. 019.
 18. Liu W, Guo Y, Zhao M, Li H, Zhang Z. Ring-oven washing tech-
nique integrated paper-based immunodevice for sensitive detec-
tion of cancer biomarker. Anal Chem. 2015;87:7951–7. https:// 
doi. org/ 10. 1021/ acs. analc hem. 5b018 14.
 19. Pang B, Zhao C, Li L, Song X, Xu K, Wang J, Liu Y, Fu K, Bao 
H, Song D, Meng X, Qu X, Zhang Z, Li J. Development of a 
low-cost paper-based ELISA method for rapid Escherichia coli 
O157:H7 detection. Anal Biochem. 2018;542:58–62. https:// doi. 
org/ 10. 1016/j. ab. 2017. 11. 010.
 20. Ma L, Nilghaz A, Choi JR, Liu X, Lu X. Rapid detection of clenb-
uterol in milk using microfluidic paper-based ELISA. Food Chem. 
2018;246:437–41. https:// doi. org/ 10. 1016/j. foodc hem. 2017. 12. 022.
 21. FengS, Hua MZ, Roopesh MS, Lu X. Rapid detection of three 
mycotoxins in animal feed materials using competitive ELISA-
based origami microfluidic paper analytical device (μPAD). Anal 
Bioanal Chem. 2023;415:1943–51. https:// doi. org/ 10. 1007/ 
s00216- 023- 04612-y.
 22. Zhu X, Xiong S, Zhang J, Zhang X, Tong X, Kong S. Improving 
paper-based ELISA performance through covalent immobilization 
of antibodies. Sens Actuators B Chem. 2018;255:598–604. https:// 
doi. org/ 10. 1016/j. snb. 2017. 08. 090.
 23. Carneiro MCCG, Rodrigues LR, Moreira FTC, Sales MGF. Paper-
based ELISA for fast CA 15–3 detection in point-of-care. Micro-
chem J. 2022;181:107756. https:// doi. org/ 10. 1016/j. microc. 2022. 
107756.
 24. Vashist SK, Lam E, Hrapovic S, Male KB, Luong JH. Immo-
bilization of antibodies and enzymes on 3-aminopropyltriethox-
ysilane-functionalized bioanalytical platforms for biosensors and 
diagnostics. Chem Rev. 2014;114:11083–130. https:// doi. org/ 10. 
1021/ cr500 0943.
 25. Zhou M, Yang M, Zhou F. Paper based colorimetric biosensing 
platform utilizing cross-linked siloxane as probe. Biosens Bioelec-
tron. 2014;55:39–43. https:// doi. org/ 10. 1016/j. bios. 2013. 11. 065.
 26. Evans E, Gabriel EFM, Karlos W, Coltrob T, Garcia CD. Rational 
selection of substrates to improve color intensity and uniform-
ity on microfluidic paper-based analytical devices. Analyst. 
2014;139:2127–32. https:// doi. org/ 10. 1039/ C4AN0 0230J.
 27. Wang S, Chen Z, Choo J, Chen L. Naked-eye sensitive ELISA-
like assay based on gold-enhanced peroxidase-like immunogold 
activity. Anal Bioanal Chem. 2016;408:1015–22. https:// doi. org/ 
10. 1007/ s00216- 015- 9219-8.
 28. Yoshioka M, Mukai Y, Matsui T, Udagawa A, Funakubo H. 
Immobilization of ultra-thin layer of monoclonal antibody on 
glass surface. J Chromatogr. 1991;31(566):361–8. https:// doi. 
org/ 10. 1016/ 0378- 4347(91) 80252-8.
 29. Busa LSA, Maeki M, Ishida A, Tani H, Tokeshi M. Simple and 
sensitive colorimetric assay system for horseradishperoxidase 
using microfluidic paper-based devices. Sens Actuators B: Chem. 
2016;236:433–41. https:// doi. org/ 10. 1016/j. snb. 2016. 06. 013.
 30. Malvano R, Boniolo A, Dovis M, Zannino M. ELISA for anti-
body measurement: aspects related to data expression. J Immunol 
Methods. 1982;48:51–60. https:// doi. org/ 10. 1016/ 0022- 1759(82) 
90209-5.
 31. Blanco RM, Guisán J. Stabilization of enzymes by multipoint 
covalent attachment to agarose-aldehyde gels. Borohydride reduc-
tion of trypsin-agarose derivatives. Enzyme Microb Technol. 
1989;11:360–6. https:// doi. org/ 10. 1016/ 0141- 0229(89) 90020-3.
Publisher's Note Springer Nature remains neutral with regard to 
jurisdictional claims in published maps and institutional affiliations.