Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope

Aramesh, Morteza; Forró, Csaba; Dorwling-Carter, Livie; Lüchtefeld, Ines; Schlotter, Tilman; Sj, Ihle; Shorubalko, Ivan; Hosseini, Vahid; Momotenko, Dmitry; Zambelli, Tomaso; Klotzsch, Enrico; Vörös, János

doi:10.1038/s41565-019-0493-z

Cited by 52 publications

(55 citation statements)

References 50 publications

(51 reference statements)

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“…TEM grids on silicon scaffolds (4 × 4 mm frame) with 30 nm thick free‐standing silicon nitride windows (10 × 10 µm) were purchased from Norcada Inc., Canada. Polymers designed to minimize protein adhesion, namely, poly(acrylamide)‐g‐(PMOXA, 1,6‐hexanediamine, 3‐amino‐propyldimethylsilanol) in short PAcrAm‐g‐PMOXA and poly(acrylamide)‐g‐(PEG, 1,6‐hexanediamine, 3‐amino‐propyldimethylsilanol) in short PAcrAm‐g‐PEG were obtained from SuSoS AG, Switzerland, [ 42,43,56 ] and a poly(dimethyl acrylamide) copolymer with glycidyl methacrylate (GMA) in short copoly(DMA‐GMA‐MAPS) was synthesized by the Istituto di Scienze e Tecnologie Chimiche (SCITEC), Italy. Antigen‐binding fragment (Fab) of anti‐biotin IgG (800‐101‐098) was purchased from Rockland Immunochemicals.…”

Section: Methodsmentioning

confidence: 99%

“…recently used PAcrAm‐g‐PMOXA to coat solid‐state nanopores that were located close to the tip of an atomic force microscope (AFM) and these nanopore tips made it possible to sense secreted molecules on the surface of living cells in culture without clogging. [ 43 ] Zilio et al. used copoly(DMA‐GMA‐MAPS), to minimize protein adsorption onto thermoplastics.…”

Section: Introductionmentioning

confidence: 99%

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Polymer Coatings to Minimize Protein Adsorption in Solid‐State Nanopores

et al. 2020

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can be classified as biological, [8] solidstate, [9] or a combination of the two. [10] Biological nanopores include channel proteins such as the product of Curli specific gene G (CsgG) from Escherichia coli, [11] α-hemolysin (αHL) from Staphylococcus aureus, [12] Mycobacterium smegmatis porin A (MspA) [13] and Aeromonas hydrophila Aerolysin (AeL), [14] which spontaneously embed themselves in a lipid bilayer. These types of nanopores can be produced in large numbers by protein expression and purification, have well-characterized crystal structures, and have played a critical role in advancing the resistive pulse sensing technique. [15-17] It is, however, difficult to generate protein pores with diameters that exceed 4 nm. Another limitation is the need to reconstitute these protein pores into lipid or block copolymer membranes, which can be mechanically and chemically fragile. [18] Conversely, solid-state nanopores can be fabricated in various materials such as silicon nitride, silicon oxide, aluminum oxide, hafnium oxide, or graphene. [9,19] Their diameters and geometries can be tuned depending on user requirements, and they can be used repeatedly for experiments with considerable experimental flexibility (e.g., temperature, buffer conditions, presence of detergents or solvents, extreme pH values, applied potential differences, etc.). [20-22] Despite these attractive characteristics, solid-state nanopores suffer from at least three drawbacks. First, they usually interact nonspecifically with biomolecules, leading to resistive pulse artifacts such as attenuated rotation, translation and Nanopore-based resistive-pulse recordings represent a promising approach for single-molecule biophysics with applications ranging from rapid DNA and RNA sequencing to "fingerprinting" proteins. Based on advances in fabrication methods, solid-state nanopores are increasingly providing an alternative to proteinaceous nanopores from living organisms; their widespread adoption is, however, slowed by nonspecific interactions between biomolecules and pore walls, which can cause artifacts and pore clogging. Although efforts to minimize these interactions by tailoring surface chemistry using various physisorbed or chemisorbed coatings have made progress, a straightforward, robust, and effective coating method is needed to improve the robustness of nanopore recordings. Here, covalently attached nanopore surface coatings are prepared from three different polymers using a straightforward "dip and rinse" approach and compared to each other regarding their ability to minimize nonspecific interactions with proteins. It is demonstrated that polymer coatings approach the performance of fluid lipid coatings with respect to minimizing these interactions. Moreover, these polymer coatings enable accurate estimates of the volumes and spheroidal shapes of freely translocating proteins; uncoated or inadequately coated solid-state pores do not have this capability. In addition, these polymer coatings impart physical and chemical stability and enable effic...

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Polymer Coatings to Minimize Protein Adsorption in Solid‐State Nanopores

et al. 2020

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“…Different kinds of proteins, [97,98] DNA-protein complexes, [99,100] biomarkers, enzyme flexibilities, [101] and biochemical processes [5] have been widely studied through the electrical signals obtained from nanopores. Now, with the help of high-sensitivity and specific optical signals, more detailed research on those biochemical processes and molecules in suit can be conducted.…”

Section: Identification Of Complexes and Biochemical Processes At Thementioning

confidence: 99%

Electro‐Optical Detection of Single Molecules Based on Solid‐State Nanopores

Tong

et al. 2020

Small Structures

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Solid-state nanopore-based biosensors have received considerable attention in the past two decades and are promising for the next-generation single-molecule detection and sequencing platforms. Typically, the sensor is comprised of a thin inorganic membrane that separates two chambers containing an ion conductive solution. A nanoscale pore is drilled in the membrane as the only channel connecting the two chambers (cis chamber and trans chamber). The charged molecules are driven through the nanopore by an electric field applied across the membrane. At the same time, as the molecules block a fraction of the ionic current that flows through the pore, there will be a set of current pulses. The characteristics of the pulses, such as dwell time (duration of the pulses) and current blockages, give us a wealth of information about the passing analytes. Typically, excluded volumes, charges, capture rates, configurations, dynamics, and possible interactions of the analyte molecules can be concluded from electrical signals. Biological nanopores have been commercially used for DNA sequencing. [1] However, for solid-state nanopores, there are still challenges that need to be addressed for commercial use: specifically, spatial resolution, temporal resolution, and reproducibility. To achieve the identification of nucleotides during a stepwise displacement of DNA through the nanopores, our sensors must have a spatial resolution of 0.34 nm, considering the distance between adjacent base pairs of a double-stranded DNA chain. [2] Such a spatial resolution is a real challenge for nanofabrication of solid-state pores. Temporal resolution depends on the maximum bandwidth of the platforms and translocation speed of the analyte molecules. Limited temporal resolution leads to anomalously low event rates and distorted signals of proteins. [3] Signal reproducibility is another problem for solid-state nanopores compared with their biological counterparts. Biological nanopores, such as α-hemolysin [4] and mycobacterium smegmatis porin A, [5] serve as reliable sensors with excellent signal reproducibility because of the same genetically encoded structure. In comparison, due to the nonuniformity of the fabrication process or other unknown reasons, each solid-state nanopore is slightly different from one to another. In addition, there are other limitations for conventional solid-state nanopore sensors, such as low throughput, background noise, and lack of external handles to manipulate the analytes. Several strategies have been proposed to address these challenges, among which the combination of solid-state nanopores and optical detection has obvious advantages. Introducing a synchronized optical detection would help to improve the spatial resolution of solid-state nanopore platforms. Optical detection, in particular fluorescence [6] and surface-enhanced Raman spectroscopy (SERS), [7,8] has been widely used for

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“…This was done by construction of a nanopore with diameter as small as 5 ± 1 nm onto the flattened apex of FluidFM probe, so called force-controlled scanning nanopore microscope. By this means, an integration of solid-state nanopore and AFM was accomplished, and it can be used to stochastically sense the secreted molecules, and the activity of ion channels in arbitrary locations both inside and outside a cell (Aramesh et al, 2019). Besides surface imaging, the detection and delivery of biomolecules on-demand, the introduced system also facilitates flexibility and controllable mechanical engagement with the target samples.…”

Section: Nanoneedle For Single-cell Samplingmentioning

confidence: 99%

The Most Recent Advances in the Application of Nano-Structures/Nano-Materials for Single-Cell Sampling

Jia

Chen

2020

Front. Chem.

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The research in endogenous biomolecules from a single cell has grown rapidly in recent years since it is critical for dissecting and scrutinizing the complexity of heterogeneous tissues, especially under pathological conditions, and it is also of key importance to understand the biological processes and cellular responses to various perturbations without the limitation of population averaging. Although conventional techniques, such as micromanipulation or cell sorting methods, are already used along with subsequent molecular examinations, it remains a big challenge to develop new approaches to manipulate and directly extract small quantities of cytosol from single living cells. In this sense, nanostructure or nanomaterial may play a critical role in overcoming these challenges in cellular manipulation and extraction of very small quantities of cells, and provide a powerful alternative to conventional techniques. Since the nanostructures or nanomaterial could build channels between intracellular and extracellular components across cell membrane, through which cytosol could be pumped out and transferred to downstream analyses. In this review, we will first brief the traditional methods for single cell analyses, and then shift our focus to some most promising methods for single-cell sampling with nanostructures, such as glass nanopipette, nanostraw, carbon nanotube probes and other nanomaterial. In this context, particular attentions will be paid to their principles, preparations, operations, superiorities and drawbacks, and meanwhile the great potential of nano-materials for single-cell sampling will also be highlighted and prospected.

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Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope

Cited by 52 publications

References 50 publications

Polymer Coatings to Minimize Protein Adsorption in Solid‐State Nanopores

Polymer Coatings to Minimize Protein Adsorption in Solid‐State Nanopores

Electro‐Optical Detection of Single Molecules Based on Solid‐State Nanopores

The Most Recent Advances in the Application of Nano-Structures/Nano-Materials for Single-Cell Sampling

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