96-well microtiter plates, widely used in immunoassays, face challenges such as prolonged assay time and limited sensitivity due to the lack of analyte transport control. Orbital shakers, commonly employed to facilitate mass transport, offer limited improvements and can introduce assay inconsistencies. While microfluidic devices offer performance enhancements, their complexity and incompatibility with existing platforms limit their wide adoption. This study introduces a novel microfluidic 96-well cover designed to convert a standard 96-well plate to a mass-transport-controlled surface bioreactor. The cover employs microfluidic methods to enhance the diffusion flux of analytes toward the receptors immobilized on the well bottom. Both simulation and experimental results demonstrated that the cover significantly enhances the capture rate of analyte molecules, resulting in increased signal strength for various detection methods and a lower detection limit. The cover serves as an effective add-on to standard 96-well plates, offering enhanced assay performance without requiring modifications to existing infrastructure or reagents. This innovation holds promise for improving the efficiency and reliability of microtiter plate based immunoassays.

1.
J. P.
Gosling
, “
A decade of development in immunoassay methodology
,”
Clin. Chem.
36
(
8
),
1408
1427
(
1990
).
2.
S.
Souf
, “
Recent advances in diagnostic testing for viral infections
,”
Biosci. Horiz.
9
,
hzw010
(
2016
).
3.
K.
Rajewsky
, “
Clonal selection and learning in the antibody system
,”
Nature
381
(
6585
),
751
758
(
1996
).
4.
J.
Weber
,
H.
Peng
, and
C.
Rader
, “
From rabbit antibody repertoires to rabbit monoclonal antibodies
,”
Exp. Mol. Med.
49
(
3
),
e305
(
2017
).
5.
W. C.
Cheung
,
S. A.
Beausoleil
,
X.
Zhang
,
S.
Sato
,
S. M.
Schieferl
,
J. S.
Wieler
,
J. G.
Beaudet
,
R. K.
Ramenani
,
L.
Popova
,
M. J.
Comb
,
J.
Rush
, and
R. D.
Polakiewicz
, “
A proteomics approach for the identification and cloning of monoclonal antibodies from serum
,”
Nat. Biotechnol.
30
(
5
),
447
452
(
2012
).
6.
F.
Kong
,
L.
Yuan
,
Y. F.
Zheng
, and
W.
Chen
, “
Automatic liquid handling for life science: A critical review of the current state of the art
,”
J. Lab. Autom.
17
(
3
),
169
185
(
2012
).
7.
M. A.
Torres-Acosta
,
G. J.
Lye
, and
D.
Dikicioglu
, “
Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices
,”
Biochem. Eng. J.
188
,
108713
(
2022
).
8.
E.
Engvall
and
P.
Perlmann
, “
Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G
,”
Immunochemistry
8
(
9
),
871
874
(
1971
).
9.
I.
Weeks
,
I.
Beheshti
,
F.
McCapra
,
A. K.
Campbell
, and
J. S.
Woodhead
, “
Acridinium esters as high-specific-activity labels in immunoassay
,”
Clin. Chem.
29
(
8
),
1474
1479
(
1983
).
10.
W. B.
Dandliker
,
R. J.
Kelly
,
J.
Dandliker
,
J.
Farquhar
, and
J.
Levin
, “
Fluorescence polarization immunoassay. Theory and experimental method
,”
Immunochemistry
10
(
4
),
219
227
(
1973
).
11.
J. S.
Rossier
and
H. H.
Girault
, “
Enzyme linked immunosorbent assay on a microchip with electrochemical detection
,”
Lab Chip
1
(
2
),
153
157
(
2001
).
12.
M. A.
Lancaster
and
J. A.
Knoblich
, “
Generation of cerebral organoids from human pluripotent stem cells
,”
Nat. Protoc.
9
(
10
),
2329
2340
(
2014
).
13.
I.
Pereiro
,
A.
Fomitcheva-Khartchenko
, and
G. V.
Kaigala
, “
Shake it or shrink it: Mass transport and kinetics in surface bioassays using agitation and microfluidics
,”
Anal. Chem.
92
(
15
),
10187
10195
(
2020
).
14.
C.-C.
Lin
,
J.-H.
Wang
,
H.-W.
Wu
, and
G.-B.
Lee
, “
Microfluidic immunoassays
,”
SLAS Technol.
15
(
3
),
253
274
(
2010
).
15.
G. M.
Whitesides
, “
The origins and the future of microfluidics
,”
Nature
442
(
7101
),
368
373
(
2006
).
16.
K. N.
Han
,
C. A.
Li
, and
G. H.
Seong
, “
Microfluidic chips for immunoassays
,”
Annu. Rev. Anal. Chem.
6
(
1
),
119
141
(
2013
).
17.
P.
Yager
,
T.
Edwards
,
E.
Fu
,
K.
Helton
,
K.
Nelson
,
M. R.
Tam
, and
B. H.
Weigl
, “
Microfluidic diagnostic technologies for global public health
,”
Nature
442
(
7101
),
412
418
(
2006
).
18.
C. D.
Chin
,
T.
Laksanasopin
,
Y. K.
Cheung
,
D.
Steinmiller
,
V.
Linder
,
H.
Parsa
,
J.
Wang
,
H.
Moore
,
R.
Rouse
,
G.
Umviligihozo
,
E.
Karita
,
L.
Mwambarangwe
,
S. L.
Braunstein
,
J.
van de Wijgert
,
R.
Sahabo
,
J. E.
Justman
,
W.
El-Sadr
, and
S. K.
Sia
, “
Microfluidics-based diagnostics of infectious diseases in the developing world
,”
Nat. Med.
17
(
8
),
1015
1019
(
2011
).
19.
L.
Mou
and
X.
Jiang
, “
Materials for microfluidic immunoassays: A review
,”
Adv. Healthcare Mater.
6
(
15
),
1601403
(
2017
).
20.
I.
Pereiro
,
J. F.
Cors
,
S.
Pané
,
B. J.
Nelson
, and
G. V.
Kaigala
, “
Underpinning transport phenomena for the patterning of biomolecules
,”
Chem. Soc. Rev.
48
(
5
),
1236
1254
(
2019
).
21.
A. H. C.
Ng
,
R.
Fobel
,
C.
Fobel
,
J.
Lamanna
,
D. G.
Rackus
,
A.
Summers
,
C.
Dixon
,
M. D. M.
Dryden
,
C.
Lam
,
M.
Ho
,
N. S.
Mufti
,
V.
Lee
,
M. A. M.
Asri
,
E. A.
Sykes
,
M. D.
Chamberlain
,
R.
Joseph
,
M.
Ope
,
H. M.
Scobie
,
A.
Knipes
,
P. A.
Rota
,
N.
Marano
,
P. M.
Chege
,
M.
Njuguna
,
R.
Nzunza
,
N.
Kisangau
,
J.
Kiogora
,
M.
Karuingi
,
J. W.
Burton
,
P.
Borus
,
E.
Lam
, and
A. R.
Wheeler
, “
A digital microfluidic system for serological immunoassays in remote settings
,”
Sci. Transl. Med.
10
(
438
),
eaar6076
(
2018
).
22.
B.
Li
,
L.
Li
,
A.
Guan
,
Q.
Dong
,
K.
Ruan
,
R.
Hu
, and
Z.
Li
, “
A smartphone controlled handheld microfluidic liquid handling system
,”
Lab Chip
14
(
20
),
4085
4092
(
2014
).
23.
E.
Engvall
, “
The ELISA, enzyme-linked immunosorbent assay
,”
Clin. Chem.
56
(
2
),
319
320
(
2010
).
24.
T. M.
Squires
,
R. J.
Messinger
, and
S. R.
Manalis
, “
Making it stick: Convection, reaction and diffusion in surface-based biosensors
,”
Nat. Biotechnol.
26
(
4
),
417
426
(
2008
).
25.
L.
Chaiet
and
F. J.
Wolf
, “
The properties of streptavidin, a biotin-binding protein produced by streptomycetes
,”
Arch. Biochem. Biophys.
106
,
1
5
(
1964
).
26.
C. M.
Dundas
,
D.
Demonte
, and
S.
Park
, “
Streptavidin–biotin technology: Improvements and innovations in chemical and biological applications
,”
Appl. Microbiol. Biotechnol.
97
(
21
),
9343
9353
(
2013
).
27.
R. F.
Delgadillo
,
T. C.
Mueser
,
K.
Zaleta-Rivera
,
K. A.
Carnes
,
J.
González-Valdez
, and
L. J.
Parkhurst
, “
Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin
,”
PLoS One
14
(
2
),
e0204194
(
2019
).
28.
M.
Kunitz
,
M. L.
Anson
, and
J. H.
Northrop
, “
Molecular weight, molecular volume, and hydration of proteins in solution
,”
J. Gen. Physiol.
17
(
3
),
365
373
(
1934
).
29.
S.
Wang
,
B.
Li
,
D.
McLeod
, and
Z.
Li
, “
A handheld plug-and-play microfluidic liquid handling automation platform for immunoassays
,”
HardwareX
14
,
e00420
(
2023
).
30.
R. S.
Downen
,
Q.
Dong
,
J. L.
Chen
, and
Z.
Li
, “
Design and fabrication of a low-cost microfluidic cartridge with integrated pressure-driven check valve for molecular diagnostics platforms
,”
J. Micromech. Microeng.
33
(
11
),
115003
(
2023
).
You do not currently have access to this content.