Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor

TY - JOUR

T1 - Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor

AU - Zhang, Xialin

AU - Galenkamp, Nicole Stéphanie

AU - Heide, Nieck Jordy van der

AU - Valero Moreno, Julián

AU - Maglia, Giovanni

AU - Kjems, Jørgen

PY - 2023/5

Y1 - 2023/5

N2 - Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

AB - Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

KW - functionalization

KW - general applicability

KW - modularity

KW - nanobodies

KW - nanopores

KW - protein detection

KW - specificity

KW - Nanopores

KW - Spike Glycoprotein, Coronavirus

KW - COVID-19

KW - Proteins

KW - SARS-CoV-2

KW - Animals

KW - DNA

KW - Mice

KW - Single-Domain Antibodies/metabolism

U2 - 10.1021/acsnano.2c12733

DO - 10.1021/acsnano.2c12733

M3 - Journal article

C2 - 37127291

SN - 1936-086X

VL - 17

SP - 9167

EP - 9177

JO - ACS Nano

JF - ACS Nano

IS - 10

ER -