FIELD OF THE INVENTION

The present i nvention relates to a method for preparation of a specimen containing evenly distributed nanoparticles. Such an evenlv distributed specimen is suitable for imaaina-based observations and quantitative characterization using a transmission electron microscope (TEM). The invention facilitates real-time investigation of morphology, size, and dispersion of nanoparticles in a liquid sample, but without causing agglomerations of nanoparticles due to geometrical constraints of a specimen kit.

BACKGROUND OF THE IN VENTION

Nanoparticles or nanoparticle-based formulations offer the advantage of efficient delivery to the target tissue for enhanced therapeutic or diagnostic purposes, which is usually related to their size, shape, surface properties, and aggregation/agglomeration states. Comprehensive physicochemical characterization of nanoparticles with respect to their size distribution, aggregation / agglomeration state, and shape in aqueous or physiological environments is important, yet challenging, for their use in biomedical applications and compliance with safety regulations. The aggregation/agglomeration of nanoparticles in biological fluids plays a critical role in determining the physical size, shape, and surface properties that are crucial for biological recognition, yet an image-based observation of such aggregation/agglomeration is difficult to achieve in a solid phase. Transmission electron microscope (TEM) is a unique and powerful tool for observing nanoparticles. However, due to the uneven spatial distribution of the nanoparticles in conventional TEM specimens, which are either prepared by drying the solution of nanoparticles onto copper grids or by freezing it on a cryostage holder, the TEM is rarely employed for evaluations of spatial distributions of nanoparticles in aqueous solutions.

SUMMARY OF THE IN VENTION

The invention relates to fabrication of a microchip nanopipet with a controlled chamber width for sorting nanoparticles from a liquid samples such as a blood sample or milky phase liquid and preventing aggregation of the particles during a drying process, which enables quantitative analyses of their ag«re«ation/a«¾lomeration states and the particle concentration in an aqueous solution. This microchip is adaptable and compatible with all kinds of commercial TEM holders. Such a nanopipet proves to be a simple and convenient sampling device for TEM image-based quantitative

characteri zati on .

In one aspect, the invention relates to method for preparing a specimen for TEM nanoparticle characterization, which comprises:

(a) providing a specimen kit containing: (i) a top substrate and bottom substrate, each of the substrates having a top surface and a bottom surface and a length of LI, a width of WL and a thickness of 77, the top and the bottom substrates being transparent and substantially parallel to each other;

(ii) a first spacer and a second spacer, each having a length of JL2, a width of W2, and a thickness of h, located beneath the top substrate and sitting on the bottom substrate, the second spacer being positioned opposite to and spaced apart from the first spacer at a distance of rf, wherein the width of the spacer W2 is smaller than the width of the top substrate Wl and

(iii) non-enc!osed chamber having two ends open to the atmosphere and formed between the top and bottom substrates and between the first and he second spacer, being

characterized by ha ving a length of LI, a width of d, and a heigh t of A defined by the thickness of the spacer, wherein the height h being smaller than the diameter of a red blood cell; (Please provide re of image or schematic).

(b) loading a nanoparticle-containing liquid sample into the chamber; and

(c) drying the liquid sample within the chamber to obtain a dry specimen containing nanoparticies attached onto the bottom surface of the top substrate and the top surface of the bottom substrates.

in another aspect, the invention relates to a specimen kit as aforementioned.

In another aspect, the invention relates to a method for analyzing TE images of nanoparticies in a liquid sample, comprising: (Please quote ref figure to elucidate the following)

(a) preparing a dry specimen for TEM nanoparticle characterization according to the method as aforementi oned ,

(b) placing the dry specimen under a TEM; and

(c) observing and analyzing the TEM images of nanoprticles in the dry specimen to obtain information on aggregation, agglomeration and/or concentration of the nanoparticies in the liquid sample.

Further in another aspect, the invention relates to a system for use in analyzing TEM images of particles in a composition, comprising: (Please quote ref figure to elucidate the following)

(a) a specimen kit as aforementioned; and

(b) a computer software containing algorithms to perform the following functions:

(i) acquiring TEM images of nanoparticies and storing the image in a computer;

(ii) pre-processing the TEM images to enhance contrast, reduce noise and background;

(iii) segmenting the nanoprticles in the images from the background;

(iv) building a training database with a user interface; and (v) classifying the individual particles based on information in the training database. In another aspect, the invention relates to a dry nanopariicle-eontainlng specimen comprising nanoparticles in dry form evenly distributed within a non-enclosed chamber of a specimen kit as aforementioned; wherein the nanoparticles are loaded into the chamber as a non-dry particle form suspended in a liquid sample, and the nanoparticles in dry form evenly distributed within the chamber are attached onto the top surface of the bottom substrate and the bottom surface of the top substrate, and wherein the even distribution of the nanoparticles within the non-enclosed chamber reflects the status of the non-dry nanoparticles suspended in the liquid sample, and further wherein the dry specimen contains no cells. The top surface of the bottom substrate and the bottom surface of the top substrate forms part of the chamber's Inner surfaces (the top and bottom inner surfaces).

These and other aspects will become apparent from the following description of the preferred embodiment, taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

The accompanying drawings il lustrate one or more embodiments of the Invention and. together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1 A'-E are schematic side views (FIGs. I A-D) and a top view (FIG. IE) showing a conventional method for preparation of a microscopy specimen.

FIGs. 2A--E are schematic side views (FIGs. 2A-D) and a top view (FIG. 2E) showing a conventional method for preparation of another microscopy specimen.

FIGs. 3A-E are schematic side views (FIGs. 3A-D) and a top view (FIG. 3E) showing

preparation of a dry microscopy specimen according to the invention.

FIGs.4A --4E are schematic side views (FIG. 4A-D) and a top view (FIG. 4E) showing

preparation of a microscopy specimen according to the invention.

FIG. 5 is a schematic perspective view of a specimen kit (or a microchip nanopipet) according to one embodiment of the invention.

FIG. 6 is a schematic cross-sectional view of a specimen kit (a microchip nanopipet).

FIGS.7A-7B are schematic top view (FIG. 7 A) and cross-sectional view (FIG. 7B) of a specimen kit along line 702 according to one embodiment of the Invention.

FIGs. 8A -8B are schematic top view (FIG. 8A) and cross-sectional view (FIG. SB) of a

specimen kit along line 702 according to another embodiment of the invention. FIGS. A.--9B are schematic top view (FIG. 9A) and cross-sectional view (FIG. 9B) of a specimen kit alone line 702 according to another embodiment of the invention.

FIG. 10 shows the geometry of the window-type TEM microchip nanopi et and sampling processes for specimen preparation: (A) schematic diagram of the device, with the dimensions and materials as indicated; (B) the nanopipet acts as a filter for simple and convenient sorting of nanoparticles to prevent entry of !arger substances in the b!ood; (C) magnified schematic diagram of the chamber with a well-defined chamber width for controlling the drying processes.

FIG. 1 1 shows the drying processes and distribution of cPEGSk-G iPs on copper grids and the nanopipet; (A) TEM images of cPEG5k~GNPs in a 5% glucose solution dried on copper grids (with carbon and SiO _s films) and in the nanopipet (with an Si _s Ny~.film); (B) counted particle numbers in four individual image zones (2.0 ujn x 2.7 pm); (C) particle number percentage vs the distance to neighboring particles in the TEM images (sum of the four image zones). Scale bar is 50 nm.

FIG. 12 shows the observation of ePEG5k-GNPs in 50% diluted blood using the TEM nanopipet: (A) TEM images of the 5% glucose solution, the 50% diluted blood, and the cPEG5k-G Ps in the 50% diluted blood in the nanopipets; (B) particle number in the four nanopipets in the TEM image zones (2,0 μιπ ^χ 2.7 μηι); (C) particle number percentage vs the distance to the neighboring particles in the four repeated blood samples in the nanopipets; (D) comparison of the particle number percentage vs distance to neighboring particles for the cPEG5k-GNPs in 50% blood and in 5% glucose. Scale bar is 20 am.

FIG. 13 shows quantifi cation of the cPEG5k-GNP concentration i n blood samples using the TEM nanopipet and inductively coupled plasma-mass spectrometry (1CPMS) analyses: (A) concept for quantifying the concentration of nanoparticles usi ng the nanopipet; (B) determination of the cPEG5k-GNPs concentration in 50% diluted blood using ICPMS and nanopipet (n ~ 3). The blue Sine is the linear fitting result with a slope ~ 1.03 (CTCPMS ~ C _naJU pi _p£! t) and r ~ 0.997. (C)

Determination of the cPEG5k-GNPs concentration in whole blood samples from a single rat at t ^:::: 0.1 , 1, 3, 7, 24. and 48 h using ICPMS (n - I ) and the nanopipet (n - 3) analytical methods. No significant difference was found between the two methods using the t test (p < 0.05) and (3D) TEM images of the cPEG5k-GNPs in the whole blood of a rat. Scale bar is 20 nm.

FlGs. 14A-B are schematic top view (FIG. 1 A) and cross-sectional view (FIG. 14B) of a specimen kit along the line, showing the width of window vs. the thickness of the films (i.e., the top and bottom substrates).

FIGs. 15A-H show images before and after preprocess (FIG. 15A-D), segmentation (FIG. 1 SEPT), and particle selector for building database (FIG. 151), and classification of images (FIG. 15J). FIGs. 16A-B show the results of analyses of TEM images of ZnO and T1O ₂ particles In sunscreen lotion.

FIGs. 17A-C show the results of analyses of TEM images of CaCO? particles in milk.

FIGs. Ί8Α-Β show the results of analy ses of TEM images of CMP slurry. DETAILED DESCRIPTION OF TOE INVENTION

DEFINITIONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term Is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein Is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments gi ven in this specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, in the case of conflict, the present document, including definitions will control.

As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of given value or range.

Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be inferred if not expressly stated.

The terms "specimen kit" and "microchip nanoplpef are interchangeable.

Particle aggregation refers to formation of clusters in a colloidal suspension and represents the most frequent mechanism leading to destabi ligation of colloidal systems.

Agglomeration refers to the sticking of particles to one another or to solid surfaces. Aggregation implies strong attractive forces and is irreversible; agglomeration Is not as strong as aggregation and is more readily reversible, i.e., particles of the colloid are easier to break apart into smaller agglomerates or individual particles. Bare particles aggregate strongly. Surface-coated particles agglomerate eventually, but can be broken up readily.

As used herein, "nanoparticles" refers to particles of 1000 nm or less, and at least one dimension of the particles must be below 100 rtm.

As used herein, when a number or a range is recited, ordinary skill in the art understand that it intends to encompass an appropriate, reasonabie range for the particular field related to the invention.

By greater than 0.5 tun but less than 5 μηι it meant that all tenth and integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 0.6, 0.7, 0.8 and 1 , 2, 3. and 4 pm unit amounts are included as embodiments of this invention.

By ranging from 50 nm to 300 nm it meant that all integer unit amounts within the range are specifically disclosed as part of the invention. ^' Thus, 50, 51 , 52, . . . , 298, 299, and 300 nm unit amounts are included as embodiments of this invention.

By ranging from 5 pm to 30 μηα it meant that all integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 5, 6, 7, . . . _t 28, 29, and 30 μηι are included as embodiments of this invention.

Energy Dispersive X-Ray Analysis (EDX), referred to as EDS or ED AX, is an x-ray technique used to identify the elemental composition of materials.

Support vector machines (SVMs, also support vector networks ¹ ') are supervised learning models with associated learning algorithms that analyze data and recognize patterns, used for classification and regression analysi .

The terms "even distribution of nanoparticles" means the distance between neighboring nanoparticles exhibits an uniform Gaussian distribution as shown in FIG. VIC (sample preparation using nanopipet ). FIGs. 1 1 A-B (sample preparation using copper grid) show two distinct peaks, which represent large variations in distance between neighboring nanoparticles.

A specimen kit having a tiny chamber is disclosed for use in preparation of a specimen for TE The height of the chamber i smaller than the dimension of a blood cell and therefore i adapted to sort nanoparticles from blood cells. The specimen prepared according to the invention is suitable for TEM observations of the distribution status of nanoparticles in a liquid sample such as blood. The small height of the chamber eliminates the possibility of aggregation and/or agglomeration of the nanoparticles during drying. Thus, a specimen prepared under this invention is suitable for TEM observations of the dispersion and/or agglomeration of nanoparticles in a liquid sample.

In one aspect, the invention relates to a specimen kit (FIGs. 5, 6, 10, 14) comprising: fa) a top substrate and bottom substrate, each of the substrates having a top surface and a bottom surface and length of JUT, a width of W1, and a thickness of 77, the top and the bottom substrates being transparent and parallel to each other;

(b) a first spacer and a second spacer, each having a length of , a width of W2, and thickness of h, located beneath the top substrate and sitting on the bottom substrate, the second spacer being opposite to and spaced apart from the first spacer at a distance of (L wherein the width of the spacer W2 is smaller than the width of the top substrate Wl and

(c) a chamber formed between the top and the bottom substrates and between the first and the second spacers, the chamber having two ends open to the atmosphere and being characterized by having a length of Li, a width of d, and a height of h defined by the thickness of the spacer, wherein the height h being smaller than the diameter of a red blood ceil.

In one embodiment of the invention, the top substrate further comprises a plurality of holes or channels or slits, the size of the holes or channels or slits is adapted to be sufficiently narrow so that a liquid sample within the chamber is restricted from leaking out/slipping through the holes or channels or slits i n the top substrate.

in another embodiment of the invention, the top substrate does not contain hollow structures such as holes or slits or channels.

The bottom substrate of the specimen kit is flat and continuous without any holl ow structures formed in the bottom substrate. The chamber is not enclosed nor sealed in all sides.

The length, the width and the height of the chamber is defined by the length of the top and the bottom substrates, the distance between the two spacers, and the thickness of the spacer, respectively. Therefore, the volume of the channel is fixed and can be calculated or determined. The length of the chamber according t one embodiment of the invention is no greater than 1 cm, and the height of the chamber is smaller than the diameter of a red blood cell.

in another embodiment of the invention, the aforementioned specimen kit further comprises:

(a) a top frame having top surface and a bottom surface, the top frame being located on the top substrate and having an open window formed between the top surface and the bottom surface thereof; and

(b) a bottom frame having a top surface and a bottom surface, the bottom frame being located beneath die bottom substrate and having an open window formed between the top surface and the bottom surface thereof;

wherein each of the open windows has one window opening facing the chamber and another window opening facing away from the chamber, the window opening facing the chamber has a width not greater than 50 ηι and a length not greater than S00 urn. The top and bottom frames support the top and bottom substrates by providing strength to the substrates.

In another embodiment of the invention, the top and the bottom substrates each have thickness ranging from 50 nra to 300 nm, or 50 nm -~ 200 nra. preferably ranging from 100 nm -200 nra. It is important that the two substrates each have a thickness of no les than 50 nm to minimize deformation and to keep the top and bottom substrates (or films) in parallel during loading and drying of the liquid sample.

in another embodiment of the invention., the height of the chamber is larger than 0.1 μηι but smaller than 5 μπχ. Alternatively, the height of the chamber is smaller than or equal to 5 μ ι. in another embodiment of the invention, the height of the chamber is selected from the group consisting of less than 10 um, from 5 nm ·- 2 pm, from 5 nm - 0 5 μιη, and from 5 nm ~150 nm. in another embodiment of the invention, the chamber has a length of no greater than 1 cm. and width of smaller than 1 cm.

in another embodiment of the invention, the top and bottom, substrates each comprise a silicon nitride (SL _; N _V ) film.

in another aspect, the invention relates to a specimen kit as aforementioned for use in preparing a dry specimen for transmission electron microscopy (TEM) nanopariicle characterization.

In another aspect, the invention relates to a method for preparing a specimen for TEM

nanopariicle characterization, which comprises: (a) providing specimen kit as aforeme ti ned; and (b) loading a nanoparticle-containing liquid sample into the non-enclosed chamber. n this case, the method does not include a drying process and thus the nanoparticles in the liquid sample is observed under TEM.

Further in another aspect, the invention relates to a method for preparing a specimen for TEM nanopariicle characterization, which comprises:

(a) providing a specimen kit as aforementioned;

(b) loading a nanoparticle-containing liquid sample into the non-enclosed chamber; and

(c) drying the liquid sample within the non-enclosed chamber to obtain a dry specimen containing nanoparticles attached onto the bottom surface of the top substrate and onto the top surface of the bottom substrate.

in one embodiment of the invention, the loading step is performed by positioning one end of the chamber downward to contact with the liquid sample.

In another embodiment of the invention, the nanoparticle-containing liquid sample is at least one selected from the group consisting of a blood sample, a bodily fluid, a lotion, a chemical mechanical poSishing planarization slurry, and a calcium carbonate nanoparticle-containing liquid or milk. In another embodiment, of the invention, during drying of the liquid sample the nanoparticles are restricted from movement and aggregation and/or agglomeration.

Further in another aspect, the invention relates to a method for analyzing TEM images of nanoparticles in a liquid sample, which comprises;

(a) preparing a dry specimen for TEM nanoparticle characterization according to a method as aforementioned;

(b) placing the dr specimen under a TEM; and

(c) observing and analyzing the TEM images of nanoprtic!es in the dry specimen to obtain information on aggregation, agglomeration and/or concentration of the nanoparticles in the liquid sample.

in one embodiment of the invention, the observing and analyzing step further comprises;

(i) acquiring TEM images of the nanoparticles and storing the TEM images in a computer;

(ii ) preprocessing the TEM images to enhance contrast, reduce noi e and background

(iii) segmenting the nanoprticles in the images from the background;

(iv) building a training database with a user interface, and

(v) classifying the individual particles based on information in the training database, in another embodiment of the invention, steps (i) -~ (v) are performed with a computer software. In another embodiment of the invention, the building step comprises training the computer to recognize properties of nanoparticles. The properties may be at least one selected from the group consisting of aggregated particl es and single particles.

in another aspect, the invention relates to a system for use in analyzing TFM images of particles in a composition, wherein the system comprises:

(a) a specimen kit as aforementioned; and

(b) a computer software having algorithms to perform the following functions;

(i) acquiring TEM images of nanoparticles and storing the images in a computer;

(ii) preprocessing the TEM images to enhance contrast, reduce noise and background

(iii) segmenting the nanoprticles in the images from the background;

(iv) building a training database with a user interface; and

(v) classifying the individual particles based on information in the training database. The support vector machines (SVM) are algorithms that may be adapted to perform

preprocessing, segmenting, training database-building and particle-classification. Pre-processing step employs the following histogram equalization algorithm: .· . , 'I,

Yet in another aspect, the invention relates to a dr nanoparti de-containing specimen

characterized by an even distribution of nanopaiticles within non-enclosed for non-closed) chamber of a specimen kit as aforementioned, the dry specimen resulting from drying of a nanoparticle- containing liquid sample, wherein the even distribution of the nanopaiticles within the non-enclosed chamber reflects the status of the nanoparticles in the liquid sample. The nanoparti cl es-con tai n i ng specimen is not enclosed or is non-closed. The nanoparticie-containing liquid sample may be at least one selected from the group consisting of a blood sample, a bodily fluid, a lotion, a chemical mechanical poli shi ng/pl anarizati on slurry, and a calcium carbonate nanoparticie-containing liquid or milk.

EXAMPLES

Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

Microscopy specimen prepared by conventional methods

FIGs. 1 A-E are schematic drawings illustrating a conventional method for preparation of a microscopy specimen. A drop of biological fluid sample 102, e.g., a blood sample, which contains blood cells 104 and particles 106, e.g., nanoparticles, is placed on a piece of substrate 100 such as a copper grid (FIGs. 1 A-B, side views). The diameter of a nanoparticle is usually no greater than 1 micrometer and thus, is smaller than the diameter of a blood cell. A red blood cell (RBC) has an average diameter of 6-8 μιη, and a white blood cell (WBC) has an average diameter of 10-1.2 μ.ηι. The liquid in the biological fluid or blood sample 102 evaporates during a drying process, which causes shrinkage of the liquid drop and formation of a plurality of smaller droplets (FIG. 1 C, a side view). A surface tension 108 within each droplet drags the components, e.g., nanoparticles, therein closer and closer to each other, resulting in aggregation of the nanoparticles within the droplets. FIG. ID (a side view) illustrates two aggregation groups 1.10a and 110b formed after the liquid in the sample is completely evaporated. The aggregation of nanoparticles 110a and 110b which occur in the above prepared specimen displays a similar appearance to nanoparticle-agglonieration. which causes observation confusions between nanoparticle aggregation and nanoparticle agglomeration and gives a TE observer inaccurate or false information. FIG I E is a top view of FIG. ID, showing two aggregation groups 110a and 1 1 b formed. The microscopy specimen of FIG. i ^' E does not reflect the true status of the nanoparticles 106 in the original sample 102 because the nanoparticles 106 in the original sample 102 are evenly dispersed without any aggregation ( FIG. 1 B). This would frustrate the purpose for using a TEM to obtain information on the status, dispersion and/or

agglomeration of nanoparticles in a blood sample from subject.

FIG. 2 is another illustration of a microscopy specimen prepared according to a conventional method. A drop of biological fluid sample 102, e.g., a blood sample, which contains blood cells 104, dispersed nanoparticles 106 and nanoparticle agglomerations 21 , is placed on a piece of substrate 1 0 such, as copper grid (F!Gs, 2A-B). The liquid in the blood sample 1 2 evaporates during a drying process, which causes shrinkage of the liquid drop and formation of a plurality of smaller droplets {TIG. 2C). A surface tension 1 8 within each droplet drags the components, e.g., nanoparticles, therein closer and closer to each other, resulting in formation of aggregation 110a and 110b of the nanoparticles within the droplets (FIG. 2D). FIG. 2E is a top view of FIG. 2D, showing two aggregation groups .110» and 11 Ob, which are newly formed aggregations because they do not exist in the original blood sample 102 (FIG. 2B). Thus, the microscopy specimen prepared according to FIG. 2 would give false information to a TEM observer.

The microscopy specimen shown in FlGs. 2D-E present nanoparticle agglomeration 210, and newly formed nanoparticle aggregations 1.10a and 110b, which have similar appearance as nanoparticle agglomeration 210. The newly formed nanoparticle aggregations 1.10a and 110b are caused by a surface tension 1 8 within the droplet during the process of drying. This would frustrate the purpose for using TEM to obtain information on whether an original blood sample contains nanoparticle agglomerations or not since the microscopy specimen prepared with the conventional method does not reflect the true number of nanoparticle agglomerations.

Microscopy specimen prepared with a microchip nanopipet (i.e., a specimen kit)

This invention relates to a microcopy specimen prepared with a microchip nanoptpet that has a tiny chamber. The height of the chamber is configured to be much smaller than the diameter of a red blood cell (RBC). A RBC is smaller than a WBC. Thus, all the blood cells can be excluded/screened from entering the chamber of the microchip nanopi et. The absence of the blood cells in the specimen reduces interference with observations of nanoparticles and therefore enhances the quality and quantity examinations of the specimen. The small chamber of the microchip nanopipet holds the blood sample within, and eliminates the effect of a surface tension during a drying process. A microscopy specimen prepared according to the invention makes it possible to detect the true distribution status of the nanoparticles such as the dispersion and/or agglomeration in the original blood sample.

FIG. 3 illustrates preparation of a microscopy specimen according to the invention. A blood sample 102 is caused to contact with one end of a chamber 300 formed between a top substrate 302 and a bottom substrate 304 (FIG. 3 A) of a microchip nanopipet. The blood sample 102 contains nanoparticles 106 and b!ood ceils 104. The height h between the top substrate 302 and the bottom substrate 304 is made to be less than the diameter of a red blood ceil 104 so that all the RBCs and WBCs are excluded from entering the chamber 300. The top substrate 302 has a top surface 306 and a bottom surface 307, The bottom substrate 304 also has a top surface 3! 0 and a bottom surface 312. A height h of 10 μιη for the chamber 300 Is sufficient for TEM observations of distribution status, dispersion and/or agglomeration, of nanoparticles in a blood sample.

The nanoparticles 106 along with blood fluid enter a chamber 300 (FIG. 3B). The blood cells 104 are excluded from entering into the chamber 300 due to the cell size being bigger than the chamber's height. The liquid inside the chamber undergoes a drying process, which causes formation of a plurality of small droplets. Each droplet wraps a single nanoparticle 106 and attaches onto the Inner surfaces (i.e., the bottom surface of the top substrate and the top surface of the bottom substrate) of the chamber 300 due to an adhesion force 308 of the droplet (FIG. 3C). FIG. 3D is a side view of a microscopy specimen after the liquid in the blood sample is dried, which shows some nanoparticles 106 a ttached to the bottom surface 307 of the top substrate 302, and some nanoparticles 106 attached to the top surface 310 of the bottom substrate 304. Because of the tiny space or the small height h between the two substrates 302, 304, the number of nanoparticles 106 di stributed in a thin sample layer (H) is limited, which eliminates the possibility of aggregations of nanoparticles 106 In a thin sample layer (H). FIG. 3E is a top view of FIG, 3 D, showing that the dispersed nanoparticles 106 truly reflect a real dispersion status of the nanoparticles 106 in the original blood sample 1.02 as shown in FIG. 3B.

FIG. 4 illustrates preparation of another microscopy specimen according to the invention. A blood sample 1 2 is caused to contact with one end of a chamber 300 formed between a top substrate 302 and a bottom substrate 304 (FIG. 4A) of a microchip nanopipet. The blood sample 1 2 contains nanoparticle agglomerations 210, dispersed nanoparticles 106 and blood cells 104. The height between a top substrate 302 and a bottom substrate 304 is made to be less than the diameter of a red blood cell 104 so that all the RBCs and WBCs are excluded from entering the chamber 300. Both nanoparticles 106 and nanoparticle-agglomeration 210 enter the chamber 300 along with the fluid of the blood sample 102. Blood cells 104 cannot enter the chamber 300 due to its dimension being larger than the height of the chamber (FIG. 4B). The liquid inside the chamber undergoes a drying process, which causes formation of a plurality of small dropl ets. Each droplet wraps a single nanoparticle 106 or a group of agglomerated, nanoparticies 210 and attaches onto the Inner surfaces (i.e., the bottom surface of the top substrate and the top surface of the bottom substrate) of the chamber 300 due to an adhesion force 308 of the droplet { FIG. 4C). After the liquid withi n the chamber 300 is complete dry, some dispersed nanoparticies 106 and nanoparticle-aggiomeration 210 are attached to the bottom surface of the top substrate 302 and to the top surface of the bottom substrate 304. The tiny space or the small height h limits the number of the nanoparticies 106 and nanoparticle-aggiomeration 210 distributed in a thin sample layer (H), and hence eliminates the possibility of aggregation of the nanoparticies 106 and the nanoparticle-aggiomeration 210 a thin sample layer (H) (FIG. 4D). FIG. 4E is a top view of FIG. 4D. i which the dispersed nanoparticies 106 and nanoparticle-aggiomeration 210 of the microscopy specimen truly reflect the real situation of nanoparticies 106 and nanoparticle-aggiomeration 210 distributed in an original blood sample 102 as shown in FIG. 4B

FIG. 5 illustrates a microchip nanopipet 500 for use in preparing microscopy specimen for observations under a TEM. The microchip nanopipet 500 has a chamber 300 formed in between a top substrate 302 and a bottom substrate 304; wherei n the height of the chamber 300 is smaller than the diameter of an RBC, and the top substrate 302 is made of a material that is transparent to electrons. A chamber height of less than 10 pm is sufficient to al lo TEM observations of the distribution status of nanoparticies, and dispersion or agglomeration of nanoparticies in a blood sample 102. A first spacer 502a and a second spacer 502b are inserted between the top 302 and bottom 304 substrates to control the height of the chamber 300. The chamber 300 has a first 301a end and a second end 301b, each of the ends has an entrance 504. The entrance 504 may be configured for i nj ecti on of a sam p! e.

A top frame 506 i located on a top substrate 302, and a bottom frame 508 (which may be of the same size as the top frame) is located beneath bottom substrate 304, wherein the top frame 506 and the bottom frame 508 each have a top surface 510 and a bottom surface 51 1 , and an open window 512 formed between the top 510 and the bottom 511 surfaces, the open window 512 havi ng a height h _w equal to the thickness of the top 506 or the bottom 508 frame, and a length and a width both smaller than the length and the width of the top or bottom frame. The open window 512 receives TEM electron Beam 518 (FIGs. 5 -6).

The open window 512 in the top frame 506 and the open window 512 in the bottom frame 508 each have an opening 5.1.2c facing the chamber and another opening facing away from the chamber (FIG. I DA). The opening 512c (which faces the chamber) of the top frame open window is formed at the bottom surface of the top frame, facing toward the top substrate 302 and thus the chamber 300. The opening 512c of the bottom frame open window is formed at the top surface of the bottom frame, facing toward the bottom substrate 304 and the chamber 300. The opening 5.1.2c facing the chamber has a width 516 of no greater than 50 μηι, and a length 514 of no greater than 800 μηι. Therefore, the particle specimen within the chamber 300 may be observed through the window opening 12c (which is located at the bottom surface of the top frame and faces the top substrate 302 and the chamber 300) under a TEM from the top of the microchip nanopipet 500.

The thickness of the top and bottom substrates ranges from 50 nra to 300 nm, or 50 am ·- ^■ 200 nm, preferably in a range of 100 nm -200 nm.

The thickness of the substrates and the width of the window are highly important for use of the specimen kit in TEM image-analyses of iianoparticles according to the invention. The two substrates each .need to have a thickness of .no less than 50 am, and the window width 516c facing the chamber (i.e., the window opening that faces the chamber) needs to be less than or equal to 50pm . These requirements are necessary to minimize deformation of the top and bottom substraies and to keep the top and bottom substrates (or films) in parallel. The deformation of the substrates (or films) would result in aggregation of particles during a drying process, and incorrect TEM image analysis of nanoparticles.

FIGs. 14A-B illustrate the dimensions of the width (w) of the open window 512 c facing the chamber and the thickness of the film (the top and bottom substrates). The dimensions need to be in a certain range so that the two films (i.e., top and bottom substrates) at the portion adjacent, to (or exposed to) the open windows 512c remain parallel to each other. The width of the open window 512c facing the chamber is < 50 pm, preferable 5-30 pm, and the length is 800 pm, preferable 50- 500 urn. The thickness of the film (or substrate) is < 300 nm, preferable 50-200 nm.

FIG. 6 shows a chamber 300 formed betwee a top substrate 302 and a bottom substrate 304. An observation window 512 is made at the center of the top frame 506 of the microchip nanopipet 500. Part of the chamber 300 may be observed through the window 512 under a TEM from the top of the microchip nanopipet 500. One end of the chamber 300 serves as an entrance 504 for a liquid sample.

FIG. 7 A shows a top view over the chamber 300. A top substrate 302 is made of a flat panel and is transparen t to electrons. FIG. 7B is a cross-section view of FIG . 7 A, showing both the top substrate 302 and the bottom substrate 304, and a liquid sample, e.g., blood, 102 is filled into the chamber 300 between the substrates 30 and 304.

FIG. 8 illustrates a microchip nanopipet according to another embodiment of the invention. FIG. 8A is a top view of a top substrate 802 containing holes SOS in another microchip nanopipet, showing a pluralit of holes 803 penetrating through the top substrate 802. Observations of a specimen may be made with blood or liquid in the chamber. A better quality of observation of the specimen with a TEM may be made through the holes 803 without hindrance of the top substrate 802. FIG 8B is a cross-sectional view of FIG. SA, showing the top substrate 802. and the bottom substrate 804, a sample biood 102 filled into a chamber between the substrates 802, 804. Each hole is configured to be small enough to hold a blood sample 102 within the chamber by a surface tension 808 so that the blood sample liquid 102 does not seep through the hole 803

FIG. 9 illustrates a microchip nanopipet according to another embodiment of the invention. FIG. 9A is a top view of a top substrate 902 containing slits or channels 903 in another microchip nanopipet, showing a plurality of slits or channels (or penetrating grooves) 903 penetrating through the top substrate 902. Observations of a specimen may be made with blood or liquid in the chamber. A better quality of observation of the specimen with a TEM may be made through the slits 903 without hindrance of a top substrate 902. FIG. 9B is a cross-sectional view of FIG. 9 , showing a top substrate 902 and a bottom substrate 904, a sample blood 102 filled into a chamber between the substrates 902, 904. Each slit or channel is configured to have a width small or narrow enough to hold a blood sample 102 within the chamber by a surface tension 908 so that the blood sample liquid 102 does not seep through the slits 903.

Here, a microchip nanopipet with a narrow chamber width was constructed to prevent the aggregation of the particles during a drying process, enabling a quantitative analysis of their aggregation/agglomeration states and particle concentration in blood (FIG. 10). The upper substrate of the .nanopipet breaks the surface tension of the sample droplet, suppressing the capillary flow accompanied with evaporation of water and the aggregation of the substances when the dropl et is conventionally dried on a copper grid. This nanopipet acts as a filter for simple and convenient sorting of PEGySated gold nanoparticles in whole blood, 50% diluted blood, and 5% glucose solution. The slight aggregation of carboxyl-PEGSk-modified. gold nanoparticles (- 18%) in 50% diluted blood sample was observed while they were well-dispersed in a 5% glucose solution. Moreover, a consistent concentration for the cPEG5k-GNPs was obtained using the nanopipet and inductively coupled plasma-mass spectrometry (ICP S) analysis for both in vitro 50% diluted blood and in vivo whole blood.

We examined the possibility of the use of a nanopipet to obtain the aggregation/agglomeration states of particles in aqueous solutions Carboxyl-PEGSk-modified gold nanoparticles (cPEGSk- GNPs), which are known to be long circulating and in a well-dispersed colloidal form in biood, were dissolved in 5% glucose solution and used as model particles for examining and comparing the spatial distribution of the particles in TEM specimens dried in the nanopipet (with an Si*N film) and on copper grids (with either a carbon-film or an SiO^-film). An even spatial distribution of the particles was observed in the nanopipet with an Si _y -film (water contact angle:—32°), with the particle number in four randomly chosen 2.0 pm ^χ 2.7 um image zones determined to be 478, 467, 502, and 504 (FIGs. 1 IA-B). The distance of each cPBG5k-G P to the nearest adjacent particle (FIG. 1 ID) was a!so measured for all four of the 2.0 μ.ηι ^χ 2.7 prn image zones, and the particle number percentage (n/N) was plotted versus the distance to the neighboring particle (FIG. I ID). A single broad distribution peak was observed with a mean distance of 69.4 ± 21.6 nm (FIG. I I C), which is larger than the diameter of ePEGSk-GNP (-39.6 nm as observed by IBM, ~ 39.3 nm as measured by DLS), and indicates that most of the particles are separated from the neighboring particles. The higher percentage of particles distributed at d values greater than the particle diameter suggests thai most of the particles are distributed without contact and separated without aggregation. These results are in accordance with the understanding of cPEG5k.-GNPs, which are well -dispersed in 5% glucose solution, and indicate that the nanopipet can preserve the native spatial distribution of the particles and avoid aggregation/agglomeration in the sample solution. However, when the same sample solution was dried on the copper grid with a hydrophobic carbon-film (water contact angle: ~70°) and even on the grid with a hydrophilic Si Ox-film (water contact angle: ~30°), an obvious uneven spatial distribution of the particles was observed (FIGs. 1 1 A-B ). From the plot of the particle number percentage vs distance to the neighboring particle (FIG. 1 1 D), three peaks were obtained for both the carbon-film and SiGvfilm copper grids (FIG. 1 1C). Note: Figure caption in PDF file should be edited. It should be "Distance to neighboring particle... "The presence of two peaks with a distance smaller than the diameter of the cPEG5k-G Ps suggests t at the particles are vertically stacked or self-aggregated on the copper grids during the drying process (gradual evaporation of the bulk solvent), and even the hydrophilic surface modification with an SiOvfilm cannot prevent

aggregation, in another example, 300 nm polystyrene beads were dried both in nanopipets and on copper grids. The beads were well separated in the nanopipets but highly aggregated on the copper grids (carbon film and SiO* film). The nanopipet clearly otters a simple and convenient sampling device for preserving and quantifying the native aggregati on/ aggl om eraii on states of the particles, which are free of any artifacts introduced due to the sampling device.

Furthermore, we examined the possibility of the use of a nanopipet with a well-defined chamber width to sort out certain-sized particles from blood, which is an interesting physiological

environment in which the observation and characterization of nanoparticies is challenging, in addition to preserving the native spatial distribution of the particles, the defined narrow chamber structure of the nanopipet can also act as a filter for simple and convenient sorting of nanosized materials, preventing the entry of larger substances found in blood; in this case, the blood cells and platelets can be excluded in the sample loading process {FIG. 10B). Thus, only the submicrometer sized particles in blood plasma smaller than the chamber width of the nanopipet were sampled and observed by TEM. A 50% diluted blood sample was studied using this method, and some irregular- shaped nanoscale substances (5-20 nm) were observed that might be serum proteins, such as serum albumin (FIG. 12A). When cPEG5k-GNPs were spiked into the 50% diluted blood, both the gold nanoparticles (indicaied with the purple arrow) and the presumed blood proteins (indicated with the white arrow) were easily visualized, recognized, and used for image-based quantitative analysis (FIG. 12 A). Four nanopipets were used to load the particles ^" from the same 50% diluted blood sample, and a similar particle number was observed for each (FIG Ϊ2Β). The even spatial distribution of the particles and the reproducible quantitative results reveal that the nanopi et is a promising sampling device for sorting particles from blood. Moreover, the

aggregation/agglomeration states of the cPEG5k.-GNPs in the 50% diluted blood were evaluated by plotting the particle number percentage vs the distance to the neighboring particles (FIG. 12D). In FIG. 12C, four repeated trials showed that the cPEG5k-GNPs exhibited a broad peak (—80% particles) with distances to the neighbor particles that were larger than the diameter of the cPEGSk- GNPs, indicating thai most of the paiticles were well dispersed in the blood. A. small additional peak appearing near the diameter of the cPEG5k~GNPs (—39.6 nm) with particle number percentages of 21.7%, 18.4%, 15.4%, and 15.6% was also observed for all four of the repeated nanopipets. Because only a single peak was observed when the same concentration of cPEG5k-GNPs was spiked into a 5% glucose solution, the additional aggregation peak with a particle number of - 18% reveals that the 50% diluted biood induces a slight aggregation of the particles (FIG. S 2D). The results confirm that the nanopipet offers a simple and convenient sampling device for sorting nanoparticles and estimating the aggregation/agglomeration states of nanoparticles in blood with reproducible and quantitative results and can also be used for the analysis of other biological fluids of interest.

Moreover, intentionally aggregated, citrate-modified gold nanoparticles (citrate-GNPs) were examined. In 5% glucose, the citrate-GNPs showed -70% aggregation with 2-10 nanoparticles m each aggregate, while in 50% diluted blood, the extent of aggregation of the particles increased to -87%, with ~40% of the aggregates containing 1.1 100 particles in each aggregate. The nanopipet may, therefore, potentially be able to distinguish the aggregation extent of intentionally aggregated nanoparticles in different aqueous environments.

Quantifying the concentration of nanoparticles in a biological matrix is important for in vivo analysis of their absorption, distribution, metabolism, and excretion, as well as for pharmacokinetic and toxicity studies, particularly for nanoparti cles composed of elements that are abundant in the biological fluids in the body (e.g., C, H, O, N, P, Fe, Zn, and Ca) yet still remains a common challenge, in addition to preserving the native spatial distribution of the particles in order to prepare a homogeneous specimen and act as a filter for sorting particles .from blood, the fixed and well- defined narrow chamber volume of the nanopipet has an image volume (V) equal to the TEM image area multiplied by the chamber width of the nanopipet (V ^:::: L * ^· W ^x H). The particle number (N) in each TEM image was thus counted and divided by the total imaged volume to determine the particle concentration (C) in each sample solution (C ^:::: /V) (FIG, 13 A). The number of cPEG5k-GNPs in the nanopipet counted in the TEM images was then compared to the ICPMS analysis, which is the gold standard method for quantifying the concentration of metals (such as Au. Ag, Fe, etc.). The particle concentration of the cPEGSk-GNiPs counted in the nanopipets (n ^: - 3) and calculated by

ICPMS are consistent in the particle concentration range from 5 ^χ I0 ⁱ to 5 10° particles mi, in 50% diluted blood (FIG. 13B). Furthermore, ePEG5k-G Ps in 5% glucose (400 pL, 3 ^χ 10 ¹⁴ particles/mi.,) were injected intravenously in a rat. and blood samples were collected following injection (t - 0.1 , 1, 3, 7, 24. 48 h). Each whole blood sample was sorted using nanopipets (n ~ 3) and analyzed by ICPMS. FIG. 3C shows the comparable results obtained for the number of ePEG5k-GNPs counted in the nanopipets and measured by ICPMS. This experiment confirmed that the nanopipet is a simple and convenient sampling device for evaluating the concentration of nanoparticies using TEM . The success is attributed to the ultra small sample volume required (<1 itl . h and this tool may be used to analyze the particle concentration in local body fluids of interest. hi conclusion, we have constructed microchip-based nanopipet for the preparation of homogeneous specimens and the sorting of nanoparticies from biood. In addition to morphology based information, a TEM image-based quantitative method was developed for analyzing the shape, size/size distribution, aggregation/agglomeration states, and concentration of particles in aqueous environments of interest. Moreover, this nanopipet is adaptable to all TEM holders, mass producible, disposable, and convenient for sample loading and observations. A comprehensive physiol gical characterization of PEGylated gold nanoparticies, including their aggregation/agglomeration state and number of particle in a blood sample demonstrates the potential of this nanopipet device for nanoparticle characterization in biological fluids. Because the characterization is based on

observations of individual particles, this method can be easily extended to other particle-based materials, particularly for quantifying nanoparticies composed of the elements that are abundant in biological fluids (e.g., C, H, O, N, P. Fe, Zn, and Ca). In addition to observations under dry conditions, the sorted thin layer sample solution may be sealed in nanopipets and imaged in the nati e aqueous environment. In this study, therefore, we demonstrated that our nanopipet, a simple and conventional sampling device, offers the possibility of the use of TEM to quantitatively characterize the size/size distribution, shape, aggregation/agglomeration state, and particle concentration of na omaterials in various native environments of interest.

A computer software was used to analyze nanoparticle images. TEM images of the nanoparticles were acquired and stored in a computer, and then preprocessed to enhance contrast (FIG. I 5A-B) and reduce noise and background (FIG. 15C-D). The nanoprtic!es in the images were then segmented from the background (threshold image cut). The user or obsen'er then builds a training database with a user interface (particle selector to select primary particle and secondary particle (FIG. 1 5G).

Finally, the software was made to classify the individual nanoparticles as primary or secondary particles (FIG. 15 J) based on information the training database.

FIGs. 15A.-J show an original image downloaded from TEM database (A); the image contrast enhancement by the algorithm of histogram equalization (B); back ground noise intensification due to the procedure of enhancing image contrast (€); reduction of background noise level by a moving average filter, while retaining the signal intensity of particle (D); an original image downloaded from TEM database (E); isolation of signal of particle from the image by using interactive selection method (F); zoom in image of the original image (G); no obvious feature distortion (H) of isolated particles after image processing; a snapshot of the program which users used to build a training database by clicking individual particle (I) and (J). Primary particles are labeled in red dots (denoted "p" here), whereas the secondary particles are labeled with green dots (denoted Ύ' here) (I); The computer software enabled distinguishing of every particle (J).

FIG. 16A shows TEM image, size distribution and EDX analysis of ZnO nanoparticles in a sunscreen lotion. The sunscreen lotion was directly loaded into specimen kit and dried for TEM observations. FIG. 16B shows TEM images, size distribution and EDX analysis of TiC

nanoparticles in a sunscreen lotion.

FIG. 17A shows TEM images of CaCOj particles m milk. The mi lk was directly loaded into the specimen kit and dried for TEM observations. FIG. I7B shows TEM images of primary and

aggregated/agglomerated CaC(¼ particles in milk. FIG. 17C shows size distribution of summation, primary and aggregated/agglomerated CaCCh particles in milk The milk was directly loaded into the specimen kit and dried for TEM observations.

The chamber's height in the TEM specimen kit described in FIGs. 16- 1 7 was 2pm. The sample such as lotion, milk. etc. wa directly loaded into the specimen kit and dried for TEM observations.

FIG. ISA shows TEM images of abrasives in CMP slurry in aqueous environment observed by TEM/FEl/200keV, TEM/Hitachi/1 OOkeV, and STEM/FEi/30keV. FIG. 18B shows TEM images and the size distribution of summation, primary and aggregated/agglomerated Si0 ₂ abrasives in CMP slurry. The CMP slurry in FIG. Ί8Α-Β was directly loaded into the TEM specimen kit with 0.2μηι chamber width and sealed by epoxy resin for TEM observations.

The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.