Title：Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor

Journal：《ACS Nano》

IF: 17.1

Original link: https://doi.org/10.1021/acsnano.2c12733

Reporter: Yinnan Jing,  Master of Grade 2022

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.

At present, nanopore-based sensors are mainly divided into two categories. One is the protein nanopore family of biological sources, including Staphylococcus aureus α-hemolytic toxin, Mycobacterium smegmatis toxin protein and phage phi29 connector motor protein. The other is solid nanopores prepared by artificial materials, including inorganic silicon, silicon nitride, graphene and organic polymer films, which can be used to prepare nanopores.

Usually, traditional detection methods can only monitor one parameter. Unlike most traditional detection methods, nanohole sensors can simultaneously collect multiple information of the object to be measured from one measurement. With the increase of the dimension of the sensing system, nanopore technology provides higher spatial resolution, which can realize the detection of targets from the mixture components, and even realize the simultaneous detection of multiple targets. In addition to the advantages of high resolution, nanopore sensors can also be used to analyze samples in complex matrices, including real samples such as clinical serum.

1.Firstly, a ClyA nanopore is designed in this paper, which has a 16-base pair of DNA double-stranded junction at the wide end of the pore, and then the junction can be functionalized with multiple nanobodies. In theory, when the target protein binds to the nanobody, the ion flux through the nanopore will be changed, thereby inducing a distinguishable current signal to indicate protein detection.

Figure 1. Attachment of ssDNA to ClyA nanopore. (A) Side view (left) and top view (right) of the ClyA structure (PDB: 6mrt). Serine (colored purple) at position 110 was genetically mutated to cysteine to enable site-specific chemical modification. (B) Schematic model showing the conjugation strategy of attaching ssDNA to a ClyA nanopore. A 16-mer oligonucleotide, named f, is conjugated to a ClyA monomer via a maleimide-PEG4-DBCO linker, where the maleimide reacts with the −SH group on the protein and DBCO is clicked to the azide group on the oligo. ClyA-f monomers then oligomerize to a ClyA-f oligomer in the presence of 0.2% n-dodecyl-beta-maltoside (DDM) at 37°C. (C) SDS-PAGE analysis of the conjugation efficiency. Lane 1: protein ladder, lane 2: ClyA-S110C monomer, lane 3: after reaction of ClyA-S110C with maleimide-PEG4-DBCO (ClyA-DBCO), lane 4: after reaction of purified ClyA-DBCO with f-azide (ClyA-f). (D) Native polyacrylamide gel analysis of the oligomerization of ClyA-f. Lane 5: ClyA-f after oligomerization, lane 6: ClyA-S110C after oligomerization.

2.Functionalization of the ClyA nanopore with Spike nanobody Ty1 and electrical characterization of the nanopore. The results showed that the attachment of Ty1 nanobodies had no effect on the ClyA-f-Ty1 pores at a positive potential ( + 35 mV ), while the pores were partially blocked when a negative potential (−35 mV) was applied.

Figure 2. Functionalization of the ClyA nanopore with Spike nanobody Ty1 and electrical characterization of the nanopore. (A) Schematic model showing the strategy of functionalizing the ClyA nanopore with the Ty1 nanobody, where Ty1-f′ was immobilized on the ClyA-f nanopore by DNA strand hybridization. (B) I−V curves of ClyA-S110C (blue triangle), ClyA-f (black square), and ClyA-f-Ty1 (red circle) at applied potential ranging from −90 to 90 mV (three independent experiments). (C) Histogram showing conductance distribution of the ClyA-f nanopore with (red) and without (black) Ty1 nanobody molecules. The conductance was calculated using the sum of the absolute current value under 35 and −35 mV applied potential divided by the sum of the absolute voltage value (n = 22). (D) Representative current traces of ClyA-f-Ty1 under an applied potential of −20 mV. Io is the open pore current and Ib is the blocked pore current. (E) All-point histogram of the current traces shown in D, which demonstrated a well-defined distribution of the blockade signals. (F) Schematic model interpreting the reversible conformation change between blocked (left) and open (right) states of ClyA-f-Ty1 at an applied potential of −20 mV, which corresponded to the movement of one of the Ty1 nanobodies in and out of the vestibule of the pore. All of the experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

3.Different concentrations of spike proteins were used to test the response of nanopores modified by nanobodies to spike proteins. At lower concentrations ( 0 − 460 PM ), the opening probability of ClyA-f-Ty1 increased with the increase of Spike concentration in the whole range. The results show that molecular crowding can greatly enhance the capture of nanopores by macromolecules, that is, the capture of Ty1 by ClyA nanopores is significantly increased. The addition of bovine serum albumin greatly reduced the difference between the pores of the nanobody-modified ClyA pores. The polymer of nanobody significantly increased the binding affinity between Spike protein and Ty1.

Figure 3. Open probability of ClyA-f-Ty1 correlates positively with Spike trimer protein concentration. (A) Representative current traces of ClyA-f-Ty1 before and after the addition of increasing concentration of Spike trimer protein. (B) All-point histograms were displayed to show the current distribution before and after the addition of increasing concentration of Spike protein. (C) Curve regression of the open probability in the function of Spike concentrations. The curve was fitted by using the Hill−Langmuir equation Y = Bmax*Xh/(Kdh + Xh) (n = 1.3, Kd = 760.6 pM) (n = 3, the data are shown as mean ± standard deviation). (D) The schematic model showing the dynamics of the interaction between ClyA-f-Ty1 and Spike protein. Ty1 nanobodies dynamically move in and out of the ClyA nanopore under applied potential. Spike protein reversibly interacts with the Ty1 nanobodies attached on the nanopore, presumably in a multivalent fashion. The experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 μM BSA.

4.The feasibility of ClyA-f-NB22 for protein sensing was studied and tested. In summary, these results prove that the detection platform constructed in this study can detect the ability of smaller target analytes in nanopores by binding to coupled nanobodies.

Figure 4. ClyA nanopore functionalized with nanobody nb22 for the detection of muPA. (A) The crystal structure of muPA (purple) in complex with the nb22 nanobody (green) (PDB: 5LHR) in a cartoon presentation. The binding affinity of nb22 to muPA measured by SPR:51 kon = (4.6 ± 0.8) × 105 M−1 s−1, koff = (7.8 ± 2.2) × 10−5 s−1, KD = 0.2 ± 0.03 nM. (B) Representative current traces of ClyA-f-nb22 before and after adding 3 nM muPA under −15 mV applied potential. (C) Enlarged representative current traces after adding 3 nM muPA at −15 mV. The signals consisted of three blockade levels with current blocking percentages of 13.7 ± 0.1%, 34.1 ± 0.5%, and 63.6 ± 0.1%, respectively. (D) Heatmap of the blockade events observed after the addition of 3 nM muPA with the logarithm of the dwell time against current blockade percentage. (E) The schematic model demonstrated the conformation changes of ClyA-f-nb22 in response to muPA proteins. The experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 with the presence of 6 μM BSA

In this study, we constructed a universal nanopore sensor that can be easily functionalized with different nanobody recognition units, thereby enabling specific and sensitive detection of proteins. These findings indicate that the nanobody-functionalized nanopore platform can detect various proteins regardless of their size, shape, and charge. In addition, the polymerization of nanobodies on ClyA nanopores enables the detection of trimeric Spike proteins at picomolar concentrations, which means that the multivalent interaction between nanobodies and target proteins greatly improves the sensitivity of protein quantification.