WO2019024707A1 - Synthesizing metal nanoparticles on individual thiol-rich tags as single-molecule probes - Google Patents
Synthesizing metal nanoparticles on individual thiol-rich tags as single-molecule probes Download PDFInfo
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- WO2019024707A1 WO2019024707A1 PCT/CN2018/096753 CN2018096753W WO2019024707A1 WO 2019024707 A1 WO2019024707 A1 WO 2019024707A1 CN 2018096753 W CN2018096753 W CN 2018096753W WO 2019024707 A1 WO2019024707 A1 WO 2019024707A1
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- G01N33/587—Nanoparticles
Definitions
- the Au prescusor could be the Au (III) , Au (II) , Au (I) , such as HAuCl 4 , KAuBr 4 , AuCl 3 , KAu (SCN) 4 , KAu (SCN) 2 , Au (I) -thiolate, or other Au (III) /Au (I) compounds with different ligands.
- the invention provides methods and compositions relating to metal-nanoparticles directly and specifically synthesized on thiol groups of an individual polymeric tag.
- the methods use an approximate or at least partial auto-nucleation suppression mechanism (ANSM) to provide specificity and reduce noise (nucleation at non-target sites) .
- the tag is clonable, such as a cysteine-rich polypeptide, optionally expressed as a fusion protein.
- the nanoparticles are generally 1.5-6 nm or 3-20nm diameter, but can be made up to 100nm with enhancements such as disclosed herein.
- Clonable Tags for Electron Microscopy we demonstrate solution for specifically synthesizing EM-visible heavy metal nanoparticles (e.g. AuNPs) on individual thiol (e.g. cysteine) -rich tags expressed in cells by an auto-nucleation suppression mechanism.
- EM-visible heavy metal nanoparticles e.g. AuNPs
- thiol e.g. cysteine
- aldehyde-reactive residues e.g., lysine
- aldehyde-inert residues e.g., alanine
- MT ⁇ a smaller 31 amino acids variant
- AFP AFP developed from an insect antifreeze protein, tmAFP.
- AFP tags forms highly stable structures via disulfide bonds in oxidizing cellular compartments (Fig. 1c) . The design of these tags provides the formation of relatively stable structures that protect them from fixative cross-linking denaturation.
- D-P is a stronger gold chelating ligand, it can dissolve the precipitates formed in a series of 2-ME and HAuCl 4 mixtures.
- the further ligand-competing experiments with 6 amino acids for AuNPs synthesis revealed the gold chelating orders: -COOH, -NH 2 ⁇ thiol in 2-ME ⁇ thiol in cysteine ⁇ thiol in D-P.
- the Au (III) precursors are reduced to form [RS-Au (I) -SR] n monomers 35, 36 at RS - /Au (III) ⁇ 2: 1;
- the Au (III) precursors are gradually reduced by RS - to form [RS-Au (I) ] n cyclic loops or [RS-Au (I) ] n SR chains, aurophilically sticking together as large polymers at RS - /Au (III) ⁇ 2: 1 conditions.
- the polymers act as auto-nucleation cores for forming AuNPs (Fig. 1e1) ; while suppresses the formation of such polymers inhibits the auto-nucleation (Fig. 1e3) .
- MBP tubes are in the same patterns as those without protein case, but the tags are in totally different patterns; (ii) in the regular BSM conditions (tubes on the left of the dash lines) , all tubes have brown colors (indication of the formation of auto-nucleated and/or tag-nucleated AuNPs) ; (iii) among those ANSM conditions, only those tubes with cysteine-rich tags (MBP-2MT and MBP-2AFP) are in purple to brown colors (indication of the formation of tag-based AuNPs) , while those tubes without protein or with control protein are colorless (indicating no AuNPs formed) .
- the overexpressed FliG-MTn protein accumulated near the ends of the cells; there were many fewer AuNPs in the nucleoid (central) region of cells.
- the number of AuNPs in a cell was estimated to be of the same order of magnitude as the number of FliG-MTn protein molecules expressed in each cell.
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Abstract
Metal nanoparticles are directly synthesized on polymers comprising a plurality of thiol groups to form compositions useful as electron microscopy tags.
Description
Inventors: Wanzhong He, Xiumei Jin, all of Beijing, CN
Applicant/Assignee: National Institute of Biological Sciences, Beijing
Introduction
The development of genetically-encoded tags, analogous to the fluorescent proteins used in light microscopy (LM)
1, for electron microscopy (EM) would be highly valuable because it would enable for characterization of the precise localization of individual proteins in the ultrastructural cellular context. Such a development would overcome several limitations of traditional antibody-based immune-EM staining
2, including the poor antigen accessibility low labelling efficiency, loss of antigenicity, antibody performance dependency, poor three-dimensional representation of antigen distribution. Moreover, these genetically-encoded tags would also overcome several major limitations of light microscopy
3, e.g.: the inability to visualize cellular organelles that are not fluorescent labelled which needs the complementation with a correlated EM image, and unreliable to distinguish individual molecules with high density.
Genetically-encoded tags for EM have been explored in recent years
4-18; however, none of these efforts have realized single-molecule detection in cells. Tag-mediated generation of superoxide radicals (O
2
-) for polymerizing diaminobenzenidine into local precipitates for OsO
4 contrasting has been applied for staining cellular regions with highly abundant protein accumulation, but is unsuitable for single-molecule detection applications
4-10. Another technology is based on metal-binding tags, including ferritin
15 and metallothionein (MT)
11-14,
16-
18; only the latter of which has real potential for use in single-molecule detection; the iron-loading ferritin complex is considered too big to be a promising tag
15. MT is a well-known metal-binding protein that can bind various heavy metal ions (e.g., Zn
2+, Cd
2+, Cu
+, Ag
+, Au
+, Hg
2+, etc. ) through its 20 cysteine (Cys) residues
19. MT tags for EM have been tested with purified fusion proteins
11-13,
16 and complexes
16, and more recently for tagging fusion proteins in cells
14,
17,
18,
20. Mecogliano and DeRosier proposed the concept of using MT for generating EM-detectable gold clusters in 2006
12. The Risco group initially tested MT tags in E. Coli cells
14 and then extended their use to yeast cells and then mammalian cells: they incubated the live cells with AuCl or AuCl
3 salts to stain MT tags, forming ~1nm gold clusters. Several challenges arose with this direct incubation of unfixed cell approach, including the cellular toxicity of HAuCl
4 or AuCl salts, inevitable ultrastructural distortions, tag diffusion during processing, and high noise resulting from the unavoidable nonspecific staining. Specific staining of the MT tagged spindle protein arrays in fast-frozen yeast cells has been achieved
18; however, this approach failed to stain individual tags.
To implement cysteine-rich tags for EM detection in cells, several challenges must be overcome: designing fixative-resistant tags for achieving ultra-structures preservation and high labelling efficiency, delivery of gold precursors across membrane barriers, minimizing physiological disturbances, suppressing nonspecific staining noise, and generating gold agglomerations of sufficient size and density to enable highly sensitive and precise EM detection. To address these challenges, we developed an approach based on the syntheses of 2-6nm gold nanoparticles (AuNPs) on specific cysteine-rich tags in chemically fixed and fast-frozen cells. Building on the Brust-Schiffrin principle of thiolate-capped AuNPs synthesis
23, we implemented a new concept for the reliable and specific synthesis of AuNPs on native MT tags and on small engineered cysteine-rich tags, including one that lacked aldehyde-reactive residues ( “MTn” ) , a smaller variant of MTn ( “MTα” ) , and a disulfide-rich tag engineered from an insect antifreeze protein, tmAFP
25 ( “AFP” ) . We developed auto-nucleation suppression mechanism (ANSM) conditions that enable the specific synthesis of AuNPs on these tags. We can use ANSM to make even 1-3KDa cysteine-rich tags (e.g. MTα, 2.72kDa) , clearly visible under EM. We implemented the ANSM concept to synthesize AuNPs on cysteine-rich tags expressed in a variety of organisms such as E. Coli, S. pombe, and mammalian cells. This approach allowed for the precise mapping of individual protein molecules in intact cells with EM; in particular, we demonstrated the use of cysteine-rich tags for precise localization of individual proteins in the ultrastructural cellular context without the need for antibodies. Furthermore, we disclose that the technology is readily extended and applied to alterative thiol-rich substrates and alternative heavy metals.
Summary of the Invention
The invention provides methods for directly synthesizing heavy metal nanoparticles on polymers comprising a plurality of thiol groups, related compositions, including substrate-bound nanoparticles made by the disclosed methods, and various methods of use. The invention provides methods and compositions for synthesizing metal nanoparticles on individual thiol-rich tags as single-molecule probes, and resultant nanoparticle-bound tags.
In an aspect of the invention provides a single polymer molecule substrate labeled with a heavy metal nanoparticle, wherein the substrate comprises a plurality of thiol groups covalently bound to the nanoparticle. The thiol groups may be oxidized or reduced to thiolates, disulfides, etc.
In embodiments:
-the nanoparticle is capped with other thiol groups of ligands, especially other thiol groups with higher binding affinity for the nanoparticle. Exemplary ligands include 2-mercaptoethanol (2-ME) , penicillamine (D-P) , dithiothreitol (DTT) , dithiobutylamine (DBTA) , glutathione (GSH) , thiomalic acid (TMA) , 2-Mercaptoethylamine (2-MEA) , etc. The thiol ligands can be either used alone (e.g. 140mM 2-ME) or combined with another thiol ligands (e.g., a mixture of 2-ME and D-P, or DTT and D-P, GSH and D-P) . The functionality of the thiol ligand depends on its acid dissociation constant, pKa and structure. For example, if the substrate thiol groups are of cysteine residues (pKa=8.3-8.5) , one weak affinity thiol ligand (e.g.: 2-ME, pKa=9.64) combined with another strong affinity ligand, penicillamine (pKa=8.1) is especially effective for suppressing nonspecific noise and dissolving large thiolate-metal polymers;
-the substrate comprises a single metal nanoparticle;
-the substrate comprises a plurality of thiol-rich regions, each binding to a single metal nanoparticle;
-the metal is thiophilic element, such as Au, Ag, Pt, Hg, Zn, Cd, Pd, Pb and Te. The metal precursors can be in different valences states; for example, for Au the prescusor could be the Au (III) or Au (I) , such as HAuCl
4, AuCl
3, KAuBr
4, KAu (SCN)
4, Au (I) -thiolate, or other Au (III) /Au (I) compounds;
-the substrate is an inorganic polymer, such as a polysiloxane or polyphosphazene, or an organic polymer such as a polyurethane, a polyester (including aliphatics, such as polyglycolide, polylactic acid, and polyhydroxybutyrate, and aromatic/semi-aromatics like polyethylene terephthalate, polybutylene terephthalate, polytrimethylene terephthalate and polyethylene naphthalate) , a polyether (such as paraformaldehyde, polyethyleneglycol, polyproplylene glycol or polytetramethylene glycol) , etc.; or other composite polymers.
-the substrate comprises a biopolymer such as a polypeptide (including peptides and proteins) , polynucleotide or polysaccharide, or combinations thereof, such as gylycoproteins;
-the substrate is linked to a selective targeting probe, such as an antibody or binding fragment thereof, a lectin or binding fragment thereof, a high affinity binding protein or binding domain thereof, such as avidin, an enzyme or catalytic domain, such as horseradish peroxidase or alkaline phosohatase, or luminescent proteins, such as a GFP;
-the plurality is 2 or 3 or 4 or 6 or 9 to 20 or 40 or 60 or 90;
-the nanoparticle is 1.5, or 2 or 3 or 4 or 6 to 10 or 20 or 50 or 100nm;
-the nanoparticle comprises 30 or 40 or 50 or 100 to 200, 400 or 600 or 3000 metal atoms;
-the substrate is 1 or 2 or 3 or 5 to 10 or 20 or 100 or 500 kD;
-the substrate is cysteine-rich tag of a fusion protein, the tag comprising 2 or 3 or 4 or 6 or 9 to 20 or 40 or 60 or 90 cysteine residues which provide the plurality of thiol groups
-the substrate is in protonic solution (such as aqueous or methanolic, or aqueous-methanolic mixture) environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, and to promote synthesis of the nanoparticle on the thiol groups of the substrate; in embodiments, the inhibition is partial and the environment also comprises autonucleated metal separate from the substrate; in other embodments, the inhibition is effective to substantially preclude autonucleation of the metal; hence, the proximate ANSM conditions (at relative lower thiolate concentration conditions) can also be applied for tag-based metal NPs synthesis, wherein the mixture is can subsequently be separated, such as by FPLC or affinity column separation procedure;
-the substrate is in a protonic solution (such as aqueous or methanolic soluton) having a thiolate: metal cation (such as thiolate: Au3+) concentration ratio ≥k: 1, wherein k is 0.5, or 1 or 2 or 3 or 4 or 5 or 6 or 12. The concentration ration can be achieved by adjusting the concentration of metal, and/or the tuning the thiolate (RS
-) concentration via the reversible RSH acid dissociation equation: RSH = RS
-+ H
+, in which RS
-= RSH/ (1+10
(pKa-PH) ) ; for example, the RS
-concentration can be adjusted by varying with PH, concentration of initial RSH and its pKa. The RS
-concentration can also be achieved by the combination of two thiol ligands, e.g., 60mM 2-ME+30mM D-P; and/or
-the substrate is in or on a cell. In embodiments, the substrate is expressed in or on a cell or secreted by a cell. In embodiments, the substrate is attached to in or on another material, such as surface of a chip, circuit board, metal organic framework, etc.
The invention provides methods of using the disclosed compositions, including detecting the nanoparticle-bound substrate, for example by electron microscopy or by color change via metal nanoparticle enlargement (e.g. silver or gold enhancing) . Detection can be directly by eye or facilitated by instrumentation, such as ultraviolet-visible (UV-Vis) spectroscopy.
The invention also provides methods of making the nanoparticle-bound substrates, comprising the steps of selectively synthesizing the nanoparticle directly on the substrate.
In embodiments:
-the synthesis is performed in a protonic (e.g. aqueous) environment comprising an approximate or at least partial auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal; in embodiments, the inhibition is partial, the environment also comprises autonucleated metal separate from the substrate, and the method further comprises the step of separating the substrate-bound particle from autonucleated metal; in other embodments, the inhibition is effective to substantially preclude autonucleation of the metal; hence, the method can be practices in proximate ANSM conditions comprising relative low thiolate concentration for tag-based metal NPs synthesis, wherein additional separation, such as by FPLC or affinity column separation is required to isolate the subsrtate-bound nanoparticles;
-the method comprises the steps of selectively synthesizing the nanoparticle directly on the substrate and then enlarging the nanoparticle by nanoparticle enhancement; and/or
- the method comprises combining higher positive oxidation state heavy metal ions (such as Au (III) ) , a thiol ligands and a single polymer substrate comprising a plurality of thiol groups in an aqueous or other protonic environment under auto-nucleation suppression mechanism (ANSM) conditions or an approximate or at least partial ANSM conditions wherein the thiolate concentration is sufficient to inhibit autonucleation of the metal, wherein the thiolate reduces the metal to a lower positive valence state (such as Au (I) ) which selectively binds to the substrate thiol groups by ligand exchange to form a thiolate-metal cluster, optionally adding a stronger binding thiolate ligand, typically with a smaller pKa (such as penicillamine) to dissolve large thiolate-metal polymers and also block nonspecific metal binding to other groups on the substrate, and then optionally adding a strong reducing reagent (such as NaBH
4) to reduce the metal to zero valance (such as Au (0) ) , wherein the metal nucleates to the nanoparticle selectively on the substrate, and the substrate thiol groups are covalently linked to the nanoparticle, preferably wherein the higher positive valance heavy metal is the metal ion of a thiophilic element such as Au, Ag, Pt, Hg, Zn, Cd, Pd or Te. For example, the Au prescusor could be the Au (III) , Au (II) , Au (I) , such as HAuCl
4, KAuBr
4, AuCl
3, KAu (SCN)
4, KAu (SCN)
2, Au (I) -thiolate, or other Au (III) /Au (I) compounds with different ligands.
Other approximate ANSM conditions (lower concentration of thiolate, e.g., RS
-/Au ratio >0.5) can also be used for tag-based AuNPs synthesis (Fig. 1e2) , in this case these tag-based AuNPs need to be separated from those auto-nucleated polymer-based AuNPs by FPLC or other affinity column.
The invention encompasses all combinations of the particular embodiments recited herein. The methods may be practiced with all disclosed compositions including specific embodiments.
Fig. 1a Scheme for the implementation of clonable tags for electron microscopy; concept of cysteine-rich tag for protein localization in cells.
Fig. 1b –Fig. 1c Structures and amino acids sequences of cysteine-rich tags: metallothionein (MT) , and antifreeze protein (AFP) , MTn and MTα are engineered from MT.
Fig. 1 (d) . Procedures for synthesis of AuNPs on cysteine-rich tag: (i) unfolding the tag by thiol ligands; (ii) Au (I) compounds bind to cysteine residuals on the tag in (1) ligand retention mode (Cys-Au-SR) , (2) cysteine-bridged mode (Cys-Au-Cys)
37 and (3) possible polymer attached mode; (iii) exchanging with stronger d-penicillamine (D-P) ligand for suppressing noises; (iv) reducing the tag bound Au (I) to Au
0 atoms to form a AuNP on tag.
Fig. 1e. Schemes for synthesizing AuNPs on the tags with Brust-Schiffrin method (BSM) or the ANSM method: (1) At RS
-/Au (III) < 2: 1 conditions (BSM) , Au (I)
n SR
m forming large polymers, which act as auto-nucleation cores for forming AuNPs; (2) When the cysteine-rich tags added into the BSM condition, both the Au (I)
n SR
m polymers and the tags acted as nucleation cores for forming AuNPs, in approximate ANSM synthesizing conditions, the tag-based AuNPs needs to be separated from auto-nucleated polymer-based AuNPs by FPLC; (3) At RS
-/Au (III) > 2: 1 conditions (ANSM) , only forms countless tiny Au (I) SR species, there were no EM-visible AuNP formed due to the absent of large auto-nucleation cores; (4) When the tags added into the ANSM conditions, only individual cysteine-rich tags acted nucleation cores for forming AuNPs.
Fig. 1f. Two steps reducing scheme of AuNPs sunthesis with BSM, and the typical four-coordinate Au (III) , two-coordinate Au (I) compounds.
Description of Particular Embodiments of the Invention
The examples and embodiments described herein are for illustrative purposes and various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within this invention. Those skilled in the art will recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. The invention may exclude or be practiced in the absence of any compound, component, element or step which is not disclosed are required herein. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.
The invention provides methods and compositions relating to metal-nanoparticles directly and specifically synthesized on thiol groups of an individual polymeric tag. The methods use an approximate or at least partial auto-nucleation suppression mechanism (ANSM) to provide specificity and reduce noise (nucleation at non-target sites) . In embodiments, the tag is clonable, such as a cysteine-rich polypeptide, optionally expressed as a fusion protein. The nanoparticles are generally 1.5-6 nm or 3-20nm diameter, but can be made up to 100nm with enhancements such as disclosed herein.
In embodiments the cysteine-rich peptides can be either artificial synthesized or genetically expressed in cells; the methods provide high-efficient fabrication of Tag-based metal-NPs on fusion proteins as single-molecule probes, such as engineered antibodies, Protein A, Protein G, Protein A/G and other proteins, and avoid using the traditional nanoparticle conjugation; the methods can be used for directly synthesized metal-NPs on the cysteine-rich tags expressed in cells for identifying its functional localization; the probes can be used for single-molecule detection reagents for pathological diagnosis, single-molecule detection in cell, labeling tumors or specific tissues for specific diagnosis and treatments, etc.
Prototypical Examples
140mM 2-ME gold particle protocol. 10uL 2-mercaptoethanol (2-ME) were added into a centrifuge tube with 1mL DD H2O (PH 7.45) with 2.5uM proteins and incubated for 30 minutes at room temperature; next, 10uL of 50mM HAuCl4 were added into the centrifuge tube and vortexed well immediately, incubated for 30 minutes; then, 10uL of 10mM NaBH4 (fresh prepared at 4℃) was added into the centrifuge tube and vortexed immediately for gold nanoparticle synthesizing.
2-ME/D-P gold particle protocol. 4.25uL 2-ME were added into a centrifuge tube with 1mL PBS buffer (5mM PO4, PH 7.4) with 2.5uM proteins and incubated for 30 minutes at room temperature; next, 10uL of 50mM HAuCl4 were added into the centrifuge tube and vortexed, incubated for 30 minutes; then, 60uL 500mM D-P was added into the centrifuge tube and vortexed to mix well; finally, 10uL of 10mM NaBH4 (fresh prepared at 4℃) was added into the centrifuge tube and vortexed immediately for gold nanoparticle synthesizing.
Synthesizing gold nanoparticles in cells. 4.25uL of 2-ME were added into a centrifuge tube with 0.88mL PBS buffer (5mM PO4, PH 7.4) containing either chemically fixed cells or cold methanol fixed cells, mixed well and incubated for 60 minutes at room temperature; next, 10uL of 50mM HAuCl4 were added into the centrifuge tube and vortexed well immediately, incubated for 120 minutes; then, 100uL 500mM D-P was added into the centrifuge tube and incubated for 60 minutes; finally, 10uL of 10mM NaBH4 (fresh prepared at 4℃) was added into the centrifuge tube and vortexed immediately for gold nanoparticle synthesizing, after a few minutes, centrifuged at 2000-3000 rpm to separate the cells from the solution. The cell pellets were rapid frozen by high pressure freezing for further freeze-substitution fixation processing. For the cells directly cultured on sapphire disks, or yeast cells stuck on the poly-l-lysine coated sapphire disk, or agar-embedded bacteria or yeast, were processed the same way for AuNPs synthesizing except without the need of centrifuging.
Clonable Tags for Electron Microscopy. In this example, we demonstrate solution for specifically synthesizing EM-visible heavy metal nanoparticles (e.g. AuNPs) on individual thiol (e.g. cysteine) -rich tags expressed in cells by an auto-nucleation suppression mechanism. We disclose strategies for simultaneously preserving cellular ultra-structures and synthesizing NPs on individual tags in cells. This implementation provides a robust tool for EM detection of individual molecules in the cellular context without requiring antibodies, and also a strategy for high-efficient fabrication of single-molecule probes.
Design of fixative-resistant cysteine-rich tags for electron microscopy. An ideal tag would be small, fixative-resistant, sensitive, and precise for generating an EM-visible, electron-dense label in cells. We considered the known limitations of MT in EM tag applications when we designed our tags (Fig. 1b)
12: primarily the need for multiple copies of MT to form EM-detectable clusters
12-14,
16 and the reactivity of native MT tags with aldehyde-fixatives. To create more robust tags, first we engineered the 61 amino acids mouse MT-1 protein into an aldehyde-fixative-resistant variant, MTn (Fig. 1b) , by replacing the 21 aldehyde-reactive residues (e.g., lysine)
26,
27 with aldehyde-inert residues (e.g., alanine) . We also developed a smaller 31 amino acids variant, MTα, that is comprised of only the alpha domain of MTn. Additionally, to exploit the natural fixative-resistance of disulfide bonds, we employed another cysteine-rich tag, AFP developed from an insect antifreeze protein, tmAFP. AFP tags forms highly stable structures via disulfide bonds in oxidizing cellular compartments (Fig. 1c) . The design of these tags provides the formation of relatively stable structures that protect them from fixative cross-linking denaturation. Moreover, upon exposure to nucleophiles during the synthesis of AuNPs, these tags are able to unfold readily (Fig. 1d) ; this property is superior to tight-packed cysteine-rich protein regions (e.g., BSA and transferrin) that require harsh conditions to allow gold cluster formation
28,
29. To validate whether our engineered tags function as designed in cells, the genes for these tags were synthesized and used to construct tag-fused maltose-binding protein (MBP) that was expressed in E. Coli.
The auto-nucleation suppression mechanism (ANSM) for AuNPs synthesis. The Brust-Schiffrin method (BSM)
20 is widely used for synthesizing size tunable thiolate-capped gold nanoclusters. The core mechanism of BSM involves two reduction steps: first, thiol ligands ( “RSH” ) are used to reduce HAuCl
4 to form thiolate/Au (I) polymers; these polymers are then reduced by NaBH to form Au (0) nanoparticles. The size of the AuNPs can be tuned by varying the RSH-to-Au ratio
30 and by changing the pH
31 (Fig. 1e1) . According to the Henderson–Hasselbalch equation for RSH dissociation: pH=pKa+log (RS
-/RSH) , where RSH is thiol ligand (e.g., 2-ME) , pKa is the logarithmic acid dissocation constant of RSH, RS
-is thiolate anion, PH is the PH value of the. The RS
-concentration can be calculated out from the equation: RS
-= RSH/ (1+10
(pKa-pH) ) . Typical BSM reactions are performed using an RSH-to-Au ratios within a relatively narrow range (6: 1 to 1: 6) and feature a polymer-mediated auto-nucleation mechanism. Here, using a large excess of RSH to generate sufficent thiolate anions (RS
-) , we developed conditions that can fully suppress the polymer-mediated auto-nucleation formation of AuNPs. For example, no AuNPs were formed in BSM at an RS
-/Au ratio ≥ 2 (i.e., 2: 1) in aqueous solution (Fig. 1e3) . We thus developed a new strategy for synthesizing tag-specific AuNPs based on performing BSM at RS
-/Au ratio ≥ 2; this can suppress off-target (i.e., non-tagged) Au cluster formation via an auto-nucleation suppression mechanism (ANSM) (Fig. 1d-e4) . It should be noted that those approximate ANSM conditions (e.g., RS
-/Au ratio >0.5 ) can also be used for tag-based AuNPs synthesis (Fig. 1e2) , in this case these tag-based AuNPs need to be separated from those auto-nucleated polymer-based AuNPs by FPLC or other affinity column.
To examine the role of RS
-in BSM, we performed a series of experiments at neutral pH that tuned the concentrations of two RSH compounds, 2-mercaptoethanol (2-ME) and d-penicillamine (D-P) . These experiments revealed that AuNPs are only formed with RS
-/Au (III) ratios < 2; the presence of AuNPs was indicated by solution colors ranging from purple to brown) . When the RS
-/Au (III) ratio ≥ 2, no AuNPs were formed and the solutions remained colorless. These results imply that a striking transition is occurring at an RS
-/Au (III) ratio equal to 2; which fits the calculated RSH concentrations for generating 1mM RS
-, 156mM for 2-ME (pKa 9.64) at PH 7.45, 6mM and 40mM for D-P (pKa 8.1) at PH 7.4 and 6.5 respectively. In the mixtures of HAuCl
4 and 2-ME, Au (I)
nSR
m polymers formed as white precipitates under RS
-/Au (III) < 2: 1 conditions; while a clean solution formed at RS
-/Au (III) ≥2: 1 conditions. Similar results observed in the mixtures of HAuCl
4 and D-P, except the color of the precipitates appeared as light brown when RS
-/Au (III) < 2: 1. D-P is a stronger gold chelating ligand, it can dissolve the precipitates formed in a series of 2-ME and HAuCl
4 mixtures. The further ligand-competing experiments with 6 amino acids for AuNPs synthesis revealed the gold chelating orders: -COOH, -NH
2 < thiol in 2-ME < thiol in cysteine < thiol in D-P.
To examine the composition of the HAuCl
4-RSH mixtures under different RS
-/Au (III) ratio, we perform a series of MALDI-TOF examinations. The MALDI-TOF results demonstrated that the 1: 1 gold-to-thiolate compounds, [RS-Au (I) ] ]
n or [RS-Au (I) ]
nSR
32-35 (where n=2 to 8) were identified under RS
-/Au (III) < 2: 1 conditions; while the 1: 2 gold-to-thiolate compounds, [RS-Au (I) -SR]
n, (where n=2-6) , were identified under RS
-/Au (III) ≥2: 1 conditions. In the 1: 1 compounds, [RS-Au (I) ] ]
n or [RS-Au (I) ]
nSR form cyclic loops or zigzag chains, which are presumably responsible for forming the cloudy precipitates by aurophilic forces
34 (Fig. 1e1, f) ; while those 1: 2 compounds, [RS-Au (I) -SR]
n, tend to be soluable and form a clear solution (Fig. 1e3) . Such RS
-/Au (III) =2: 1 transition can be interpreted by the reducing reaction, Au (III) + 2RS
-= Au (I) + RSSR. The Au (III) precursors are reduced to form [RS-Au (I) -SR]
n monomers
35,
36 at RS
-/Au (III) ≥2: 1; The Au (III) precursors are gradually reduced by RS
-to form [RS-Au (I) ]
n cyclic loops or [RS-Au (I) ]
nSR chains, aurophilically sticking together as large polymers at RS
-/Au (III) < 2: 1 conditions. Thus, our results indicate that the polymers act as auto-nucleation cores for forming AuNPs (Fig. 1e1) ; while suppresses the formation of such polymers inhibits the auto-nucleation (Fig. 1e3) .
ANSM-based approach for synthesis of AuNPs on cysteine-rich tags. We initially tested our ANSM-based approach for the synthesis of AuNPs using purified MBP proteins containing various tags MBP-2MT and MBP-2AFP) (Fig. 1d-e) . and the control protein (MBP) . Comparing the colors we found: (i) MBP tubes are in the same patterns as those without protein case, but the tags are in totally different patterns; (ii) in the regular BSM conditions (tubes on the left of the dash lines) , all tubes have brown colors (indication of the formation of auto-nucleated and/or tag-nucleated AuNPs) ; (iii) among those ANSM conditions, only those tubes with cysteine-rich tags (MBP-2MT and MBP-2AFP) are in purple to brown colors (indication of the formation of tag-based AuNPs) , while those tubes without protein or with control protein are colorless (indicating no AuNPs formed) . The EM imaging of AuNPs formed by the D-P approach, confirmed the sizes of the tag-based AuNPs are ~2.3nm. These experiments matched our disclosed models (Fig. 1e) and confirm that our ANSM-based approach can be used for the specific synthesizing AuNPs on tags (Fig. 1e4) .
To optimize the ANSM-based approach for highly specific synthesizing stable and larger AuNPs on the tags, we selected representative model proteins for further tests. Two tight-packed cysteine-rich proteins, transferrin (TF, 16 Cys) and bovine serum albumin (BSA, 35 Cys) , and a maltose-binding protein (MBP, 2 Cys) were chosen as controls. To evaluate tag-based AuNPs synthesis, we tested four MBP variants that were fused to cysteine-rich tags with 11-40 cysteine residues: MBP-2AFP (24Cys) , MBP-2MT (40 Cys) , MBP-MTn (20 Cys) , and MBP-MTα (11 Cys) .
First, we used 140mM 2-ME at PH 7.45 to achieve the ANSM condition ( “2-ME approach” ) . All 4 tags all showed very dark brown colors in solutions, while the 3 control proteins (TF, BSA, MBP) were almost colorless; MBP had a faint blue color. The EM images clearly showed that no gold particles formed in the control samples but that the four proteins with tags formed ~5nm gold particles; MBP tended to aggregated as fuzzy blobs. We further optimized a protocol for synthesizing stable and large AuNPs on the cysteine-rich tags using the synergistic combination of 2-ME and D-P ( “2-ME+D-P approach” ) (Fig. 1e) . This 2-ME+D-P approach showed similar patterns as those of 2-ME approach, but all had relatively less intense colors; and MBP samples showed no blue color. The EM images for these proteins with the 2-ME+D-P approach for AuNPs synthesis showed that the AuNPs had average sizes ~3-4 nm, were well-dispersed, and did not aggregate. Finally, we used the optimized 2-ME+D-P protocol to test the aldehyde-resisting abilities of the tags. The color patterns for fixed and the unfixed groups were quite similar, except the 3 control proteins showing weaker brown colors caused by fixatives. The EM images confirmed that the 4 fixed tags could form AuNPs; very little background noise was present in the 3 control samples. These results show that the tags have some aldehyde-resisting capabilities at least in the folding states. Interestingly, for a given HAuCl
4 concentration, we found that the average sizes and size-distributions of the AuNPs depended primarily on the RSH type (2-ME or D-P) and the concentration of the tags, while the number of cysteine residues on a tag was relatively less influential.
We have thus validated that the 2-ME+D-P protocol can be used for the specific synthesis of EM-visible AuNPs on the tags with high stabilities and low noise. The 3 untagged control proteins (MBP, TF, BSA) did not form any AuNPs, even if they contained multiple cysteine residues, presumably due to their tight-packed structures. These results indicate that under our mild synthesizing conditions cysteine-rich native proteins will not interfere with the EM detection of labeled tags. Water-soluble thiol ligands other than 2-ME can be combined with D-P to achieve similar ANSM synthesis of AuNPs on tags.
Synthesizing AuNPs on individual fusion proteins as single-molecule probes. To test whether the 2-ME+D-P protocol can be used to make single-molecule probes for EM applications, we used the anti-GFP camel single-domain antibody (GBP) fused with MTn or 2AFP as single-molecule probes. We used E. Coli to overexpress GBP, GBP-MTn, and GBP-2AFP, purified these probes, and used two schemes to test GFP binding abilities of the GBP-tagged-fused probes. One scheme simply mixed the GBP-tagged-fused probes with GFP directly to form complexes prior to the AuNP synthesis. The second scheme synthesized the AuNPs on the GBP-tagged-fused probes prior to mixing and GFP binding. For scheme one, a 5-step concentration series of GFP samples deposited as 2μL spots on a PVDF membrane were used for testing the GFP binding. Both GBP-MTn (AuNPs) and GBP-2AFP (AuNPs) had excellent capacities for GFP binding, as indicated by the different color intensities for the dilution series spots. Only extremely light or no color was observed for the controls (GBP control, subjected to the AuNP synthesis process but lacking any cysteine-rich tag; GBP-2AFP control, not subjected to the AuNP synthesis process) . These experiments confirmed that the GBP-MTn and GBP-2AFP with AuNPs can be used as sensitive molecular probes for single-molecule detection in EM. For scheme two, after forming the GFP/GBP-MTn or GFP/GBP-2AFP complexes, later the AuNPs can be synthesized on the complexes. We further tested whether GFP-MT can be used as dual tags for correlative LM-EM by checking the fluorescence signals of the native GFP-MT and the GFP-MT synthesized with AuNPs, confirmed that the AuNPs synthesis did not alter the fluorescent brightness. Finally, we used GBP-MTn succeeded for probing GFP expressed in yeast cells. These tests demonstrated that this ANSM-based AuNPs synthesizing approach can be used for making common single-molecule probes, e.g.: fused the cysteine-rich tags to GBP, protein A/G, or other engineered antibodies.
Screening for conditions suitable for ANSM-based synthesis of AuNPs in E. coli. We next tested and optimized our ANSM-based AuNPs synthesis method to visualize tag-fused proteins over-expressed in E. coli cells. We tested E. coli cells over-expressing MBP, MBP-2AFP, MBP-2MT, MBP-MTn, or MBP-MTα and found that it was difficult to get gold precursors (e.g., HAuCl
4, AuCl, sodium gold thiomalate etc. ) to penetrate into live cells. Although the incubation of MT-expressing E. coli cells with AuCl formed countless AuNPs in periplasmic spaces in a previous report
14, but it is quite strange that the cytosolic proteins were mainly localized to the extracellular spaces if those AuNPs were really formed by the tags. At least, it implied that AuCl was not efficient to across the membrane barriers. To bypass such penetration barriers, we simply cracked the bacterial cell membranes by liquid nitrogen freezing (LN
2F) , a process that resulted in a marked reduction in the time required to perform our AuNPs synthesis protocols. About 20μL of bacteria pellet were cracked by LN
2F, then warmed up to 4℃ and re-suspended into 5mM PBS (PH 7.4) prior to ANSM synthesis of AuNPs with our optimized protocol. The colors of the five samples in both the solutions and the centrifuged pellets clearly illustrate the striking differences between the MBP (control) and the tags, and the corresponding EM images of these samples further confirm the formation of AuNPs in the samples with tags and almost no AuNPs formed in the control.
We next applied the optimized ANSM protocol to E. coli cells that were fast fixed and permeablized via treating with -60℃ methanol (-60℃ MeOH) with the aim of achieving uniform penetration and better preservation of cellular structure. Using this -60℃ MeOH method, the color of the MBP pellet was not different than the color of untreated cells, while the 4 samples with tags were uniformly dark. For example, the color of the MBP-2AFP pellet in the -60℃ MeOH sample was darker than the MBP-2AFP pellet in the LN2F sample. In the corresponding EM images, there were almost no AuNPs in the control sample, while many ~3-6nm gold particles formed in the cytoplasm of the FliG-MTn overexpressing cells treated with the -60℃ MeOH method. Almost no AuNPs were formed in the periplasmic spaces of these cells.
The overexpressed FliG-MTn protein accumulated near the ends of the cells; there were many fewer AuNPs in the nucleoid (central) region of cells. The number of AuNPs in a cell was estimated to be of the same order of magnitude as the number of FliG-MTn protein molecules expressed in each cell. We further validated the efficiency of our AuNP synthesis protocol by using EM to monitor the number of AuNPs in cells with different expression levels of the GBP-MT fusion protein (altered via different IPTG induction doses) .
Our methods are specific and efficient for synthesizing AuNPs on the tags of fusion proteins in E. Coli cells. Importantly, the cellular structures of E. coli, such as the cell wall, cell membranes, and periplasmic spaces, were well preserved by this protocol. We further introduced a chemical fixation step prior to the -60℃ MeOH processing by 30 min fixation with 0.5%glutaraldehyde, and confirmed that the structural preservation and AuNPs distribution pattern are quite similar to that of the -60℃ MeOH protocol alone.
Visualizing tags expressed in eukaryotic cells. After the optimization of the protocol in E. coli cells, we extended it to eukaryotic cells. To test whether the protocol is applicable for the specific detection of tag-fused proteins localized to targeted organelles, we choose three target proteins: Ost4, Nup124, and Sad1, which are expected to be localized, respectively, on the endoplasmic reticulum and nuclear envelope (NE) membrane, the nuclear pore complex (NPC) , and the spindle complex. All of these proteins were fused with GFP and GFP-MTn for validating their expression patterns and distributions with fluorescent light microscopy. All of the fusion proteins showed the expected localization patterns in yeast cells (Schizosaccharomyces pombe) .
We initially directly employed the optimized protocol that we developed with E. coli. The yeast cells were treated by scheme 1 (-60℃ MeOH 1-2 min) prior to ANSM-based AuNPs synthesis. The strains expressing GFP-MTn tags turned purple, both in solutions and in the corresponding centrifuged pellets, while the control strains expressing GFP tags did not change color. The corresponding EM images, demonstrated that a lot of AuNPs were distributed in the NE and cortical ER membranes in the cells expressing Ost4-GFP-MTn, with only a few AuNPs randomly-distributed as background in the control (yeast expressing Ost4-GFP) . The -60℃ MeOH fixation provided the most effective way for preserving the tags for AuNPs synthesis as indicated by the extremely high labeling density of AuNPs on the ER membranes.
Next, we tried to improve the preservation of the cellular ultrastructure by combining of the tag activity preservation with other fixation approaches, such as aldehyde fixation (Scheme 2; oxidizing 0.5%GA, cold MeOH) and high pressure freezing freeze-substitution fixation (HPF/FSF) (Scheme 3; oxidizing/FS 0.5%GA/FS rehydration) . Most cysteine-rich tags in the reducing environments such as cytosols are presumably in unfolded states in eukaryotic cells. The tags will lose their metal-binding ability when their thiol groups react with aldehyde-fixatives. To preserve the tag’s metal-binding activities, we used 3, 3'-dithiodipropionic acid (DTDPA) to oxidize the thiol groups of the tag to aldehyde-inert disulfide bonds prior to the aldehyde fixation; the disulfide bonds were reduced back to thiol groups during ANSM-based AuNPs synthesis. In the EM images of the Ost4-GFP-MTn overexpressing yeast cells processed with Scheme 2, the AuNPs can be clearly detected on outer surfaces of the cortical ER or NE membranes. In the Ost4-GFP-MTn overexpressing yeast cells processed with Scheme 3, the AuNPs were specifically concentrated along well-preserved NE membranes. Although a few random-distributed AuNPs background signals were present in both the control (Ost4-GFP) and the Ost4-GFP-MTn cells, the majority of AuNPs were observed in their expected locations. Finally, we validated that these methods are still effective for visualization of the less abundant tags in yeast cells, such as the NPC located Nup124-GFP-MTn and spindle located Sad1-GFP-MTn.
In summary, we have developed a general approach for synthesizing 2-6nm AuNPs directly on individual cysteine-rich tags as electron-dense labels for single-molecule detection with electron microscopy. We achieved specific synthesis of large AuNPs on individual cysteine-rich tags that can be easily detected under regular EM magnification, without requiring any further gold enhancement. We estimate that the ~3nm tag-localized AuNPs are composed of ~100-200 gold atoms by 2.5uM tags mixed with 0.5mM HAuCl
4. We developed suppression mechanism conditions involving RS
-/Au (III) |, and showed that this mechanism can be exploited for the highly specific synthesis of AuNPs on the tags. We validated the application of this mechanism with a series of experiments that characterized the tuning of the RS
-/Au (III) ratio, different types of RSH ligands, mass spectrometry analysis of the mixtures, and testing with representative model proteins including multiple control proteins and fusion proteins with tags. We adapted the basic ANSM AuNP synthesis protocol that we developed for purified proteins for use in single-molecule detection applications in both E. coli and S. pombe cells. By using both 2-Me and D-P, we were able to simultaneously achieve specific tag-based AuNP synthesis and suppression of nonspecific background singals, thus enabling single-molecule detection with minimal background noise. We confirmed that this method can be used to synthesize AuNPs on overexpressed tags, and to detect fusion proteins that were localized to specific organelles. We have demonstrated that this approach can achieve remarkably higher labeling efficiency than traditional immuno-EM. Additionally, our AuNp synthesis protocol uses mild reaction conditions that preserve the folding states and functions of the target proteins (Fig. 1d, e) , making it a powerful approach for the high-efficient fabrication of single-molecule probes by direct synthesis of AuNPs on commonly used fusion antibodies or other proteins. Our method is also readily applied for realizing correlative LM-EM imaging; for example, by making a chimeric protein fused two tags (GFP and as cysteine-rich tag) .
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Claims (26)
- A single polymer substrate labeled with a heavy metal nanoparticle, wherein the substrate comprises a plurality of thiol groups covalently bound to the nanoparticle.
- The substrate of a claim herein wherein the nanoparticle is capped with other thiol groups of ligands, especially other thiol groups with higher binding affinity for the nanoparticle.
- The substrate of a claim herein wherein the substrate comprises a single metal nanoparticle.
- The substrate of a claim herein wherein the substrate comprises a plurality of thiol-rich regions, each binding to a single metal nanoparticle.
- The substrate of a claim herein wherein the metal is thiophilic element selected from Au, Ag, Pt, Hg, Zn, Cd, Pd, Pb and Te.
- The substrate of a claim herein wherein the substrate is an inorganic polymer selected from polysiloxane and polyphosphazene, or an organic polymer selected from a polyurethane, a polyester and a polyether.
- The substrate of a claim herein wherein the substrate comprises a biopolymer such as a polypeptide, polynucleotide or polysaccharide, or combinations thereof.
- The substrate of a claim herein wherein the substrate is linked to a selective targeting probe selected from an antibody or binding fragment thereof, a lectin or binding fragment thereof, a high affinity binding protein or binding domain thereof, an enzyme or catalytic domain, and a luminescent protein.
- The substrate of a claim herein wherein the plurality is 2 or 3 or 4 or 6 or 9 to 20 or 40 or 60 or 90.
- The substrate of a claim herein wherein the nanoparticle is 2 or 3 or 4 or 6 to 10 or 20 or 50 or 100nm.
- The substrate of a claim herein wherein the nanoparticle comprises 30 or 40 or 50 or 100 to 200, 400 or 600 or 3000 metal atoms.
- The substrate of a claim herein wherein the substrate is 1 or 2 or 3 or 5 to 10 or 20 or 100 or 500 kD.
- The substrate of a claim herein wherein the substrate is cysteine-rich tag of a fusion protein, the tag comprising 2 or 3 or 4 or 6 or 9 to 20 or 40 or 60 or 90 cysteine residues which provide the plurality of thiol groups.
- The substrate of a claim herein wherein the substrate is in protonic solution environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, and to promote synthesis of the nanoparticle on the thiol groups of the substrate.
- The substrate of a claim herein wherein the substrate is in protonic solution environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, and to promote synthesis of the nanoparticle on the thiol groups of the substrate, wherein the inhibition is partial and the environment also comprises autonucleated metal separate from the substrate.
- The substrate of a claim herein wherein the substrate is in protonic solution environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, and to promote synthesis of the nanoparticle on the thiol groups of the substrate, wherein inhibition is effective to substantially preclude autonucleation of the metal.
- The substrate of a claim herein wherein the substrate is in a protonic solution having a thiolate: metal cation concentration ratio ≥k: 1, wherein k is 0.5 or 1 or 2 or 3 or 4 or 5 or 6 or 12.
- The substrate of a claim herein wherein the substrate is in or on a cell.
- A method of using the substrate of a claim herein comprising the steps of detecting the substrate.
- A method of using the substrate of a claim herein comprising the steps of detecting the substrate by electron microscopy or by color change via metal nanoparticle enlargement.
- A method of making the substrate of a claim herein comprising the steps of selectively synthesizing the nanoparticle directly on the substrate.
- A method of making the substrate of a claim herein comprising the steps of selectively synthesizing the nanoparticle on the substrate in an aqueous environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal.
- A method of making the substrate of a claim herein comprising the steps of selectively synthesizing the nanoparticle on the substrate in an aqueous environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, wherein the inhibition is partial, the environment also comprises autonucleated metal separate from the substrate, and the method further comprises the step of separating the substrate-bound particle from autonucleated metal.
- A method of making the substrate of a claim herein comprising the steps of selectively synthesizing the nanoparticle on the substrate in an aqueous environment comprising auto-nucleation suppression mechanism (ANSM) conditions comprising a thiolate concentration sufficient to inhibit autonucleation of the metal, wherein inhibition is effective to substantially preclude autonucleation of the metal.
- A method of making the substrate of a claim herein comprising the steps of selectively synthesizing the nanoparticle directly on the substrate and then enlarging the nanoparticle by nanoparticle enhancement.
- A method of making the substrate of a claim herein, comprising the step (s) of:combining higher positive oxidation state heavy metal ions, a thiol ligands and a single polymer substrate comprising a plurality of thiol groups in an aqueous or other protonic environment under auto-nucleation suppression mechanism (ANSM) conditions wherein the thiolate concentration is sufficient to inhibit autonucleation of the metal, wherein the thiolate reduces the metal to a lower positive valence state which selectively binds to the substrate thiol groups by ligand exchange to form a thiolate-metal cluster, optionally adding a stronger binding thiolate ligand to dissolve large thiolate-metal polymers and block nonspecific metal binding to other groups on the substrate, and then optionally adding a strong reducing reagent to reduce the metal to zero valance, wherein the metal nucleates to the nanoparticle selectively on the substrate, and the substrate thiol groups are covalently linked to the nanoparticle, preferably wherein the higher positive valance heavy metal is the metal ion of a thiophilic element such as Au, Ag, Pt, Hg, Zn, Cd, Pd or Te.
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