Electrophoresis device with minimal autofluorescence enabling in situ gel processing

Abstract

An electrophoresis apparatus with minimal autofluorescence enabling in-situ gel processing, and a method for manufacturing and using the electrophoresis apparatus. An exemplary electrophoresis apparatus may include a housing defining a cavity between a first and a second pane of a double-layered observation window, and may further include a plate-like gel located within the cavity. The observation window may be transparent to ultraviolet light driving derivatization reactions in the plate-like gel, and in the absence of the plate-like gel, may define window autofluorescence induced by ultraviolet light irradiation. Under the same unit area and the same ultraviolet light irradiation, the window autofluorescence per unit area may be less than 5 times that of the plate-like gel autofluorescence.

Description

[0001] Prior cross-reference

[0002] This application is based on and claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 057,248, filed July 27, 2020, and is incorporated herein by reference in its entirety for all purposes.

[0003] introduction

[0004] Polyacrylamide gel electrophoresis (PAGE) is a widely used laboratory technique for separating proteins in a sample based on their molecular weight. In a typical PAGE, a polyacrylamide gel is formed by polymerizing acrylamide between a pair of plates separated by lateral spacers and forming wells in the presence of combs. The gel typically contains a denaturing agent, sodium dodecyl sulfate (SDS), to denature the proteins and give each protein a charge proportional to its molecular weight. A sample containing proteins is loaded into the wells, and then an electric potential is applied to move the proteins from the wells along the individual gel channels. Within a given channel, proteins move at different speeds according to their size, separating proteins of different molecular weights from each other. After electrophoresis, one or both plates are removed, allowing for further processing of the gel (or the proteins within it), such as staining the proteins present with visible staining agents, imaging, and so on.

[0005] A stain-free protein fluorescence detection procedure has been developed. In this procedure, a modifying agent is added to the gel before or during electrophoresis. This modifying agent is non-fluorescent and therefore can be uniformly distributed throughout the gel without significantly increasing the background. However, irradiating the gel with ultraviolet light after electrophoresis causes the modifying agent to react locally with tryptophan residues of the protein within the gel. This reaction forms a derivatized protein containing the modified tryptophan residues, which emits light with a peak in the visible spectrum when excited with ultraviolet light. The visible light emitted by the derivatized protein can be detected, thus producing a fluorescent image of the gel. To prevent interference from the plate in the labeling and imaging steps, at least one plate is typically removed before these steps are performed.

[0006] summary

[0007] This invention provides an electrophoresis apparatus with minimal autofluorescence enabling in-situ gel processing, and a method for manufacturing and using the electrophoresis apparatus. An exemplary electrophoresis apparatus may include a housing defining a cavity between a first and a second pane of a double-paned observation window, and may further include a slab gel located within the cavity. The observation window may be transparent to ultraviolet light driving derivatization reactions in the slab gel, and in the absence of the slab gel, it may define window autofluorescence induced by ultraviolet light irradiation. Under the same unit area and the same ultraviolet light irradiation, the window autofluorescence per unit area may be less than five times that of the gel autofluorescence of the slab gel.

[0008] Brief description of the attached figures

[0009] Figure 1 This is a view of an exemplary electrophoresis apparatus, comprising a housing defining a planar cavity and a double-layered observation window, a polyacrylamide plate-like gel located in the planar cavity between the window panes of the observation window, comb teeth forming pores at the upper end of the plate-like gel, and a sealing member preventing leakage from the housing during gel formation, wherein the observation window is transparent to ultraviolet light (UV) and has minimal UV-induced autofluorescence, thereby allowing UV irradiation of the plate-like gel and detection of UV-induced fluorescence emitted by fluorescent substances in the gel, while the plate-like gel remains within the planar cavity of the housing.

[0010] Figure 2 yes Figure 1 An exploded view of the electrophoresis apparatus without comb teeth and sealing components.

[0011] Figure 3 yes Figure 1 An orthogonal front view of an electrophoresis apparatus, in which plate-like gels are indicated by dashed lines.

[0012] Figure 4 yes Figure 1 Another orthogonal front view of the electrophoresis apparatus without plate-like gels illustrates exemplary locations of polymer injection defined by discrete components of the housing.

[0013] Figure 5 yes Figure 1 The electrophoresis apparatus, without the presence of a plate-like gel, is roughly along... Figure 3 A horizontal cross-section of line 5-5.

[0014] Figure 6 yes Figure 1 The electrophoresis apparatus, without the presence of a plate-like gel, is roughly along... Figure 3Vertical cross-section of line 6-6.

[0015] Figure 7 yes Figure 1 A partial cross-sectional view of the electrophoresis apparatus, roughly taken from... Figure 6 Near the area indicated by "7" in the middle.

[0016] Figure 8 It is a flowchart that lists exemplary steps that can be performed in any suitable order and combination in a method for analyzing samples using an electrophoresis apparatus, wherein gel processing is performed in situ.

[0017] Figure 9 It is a flowchart that lists exemplary steps that can be performed in any suitable order and combination in a method for forming an electrophoresis apparatus with low UV-induced autofluorescence to achieve in situ gel processing.

[0018] Figure 10 This is an exemplary fluorescence image of an electrophoresis apparatus comprising a housing and a plate-like gel, taken after proteins have been electrophoresed in channels of the plate-like gel and derivatized under ultraviolet light to produce fluorescence emission peaks in the visible spectrum, wherein the housing defines an observation window that is ultraviolet-transparent and has minimal autofluorescence to achieve in-situ gel processing.

[0019] Figure 11 It is used with Figure 10 The contrasting fluorescence images, except that the use of a box with a viewing window opaque to ultraviolet light prevents the visualization of the fluorescently derivatized protein bands, are roughly the same as those shown. Figure 10 same.

[0020] Figure 12 These are fluorescence images of seven different acrylate (AG) samples taken under ultraviolet light irradiation to induce autofluorescence.

[0021] Figure 13 It is listed Figure 12 A table showing the relative levels of autofluorescence in seven different acrylate samples. Detailed Implementation

[0022] This invention provides an electrophoresis apparatus with minimal autofluorescence enabling in-situ gel processing, and a method for manufacturing and using the electrophoresis apparatus. An exemplary electrophoresis apparatus may include a housing defining a cavity between a first and a second pane of a double-paned observation window, and may further include a plate-like gel located within the cavity. The observation window may be transparent to ultraviolet light driving protein derivatization reactions in the plate-like gel, and in the absence of the plate-like gel, it may define window autofluorescence induced by ultraviolet light irradiation. Under the same unit area and the same ultraviolet light irradiation, the window autofluorescence per unit area may be less than five times that of the gel autofluorescence of the plate-like gel.

[0023] The inventors have discovered that using ultraviolet-transparent polymers to form the plates of an electrophoresis apparatus is insufficient for successful in-situ (i.e., without removing at least one plate) stain-free fluorescence imaging. Detection sensitivity is very low; even abundant proteins often go undetected. However, if the ultraviolet-transparent polymer also possesses minimal autofluorescence, the detection sensitivity is much higher, allowing both high-abundance and low-abundance proteins to be detected.

[0024] The inventors have also discovered that polymer injection sites of injection-molded components in electrophoresis apparatus generate increased autofluorescence, interfering with the detection of fluorescent protein derivatizations. This invention provides an improved electrophoresis apparatus having a housing in which the polymer injection sites of each injection-molded component are positioned away from the observation window, thereby reducing or eliminating interference between the individual polymer injection sites and the separated portions of the gel. The polymer injection sites defined by each injection-molded component may exist within the housing at a location offset from the observation window, or may be physically removed (e.g., cut or disconnected), thus preventing the polymer injection sites defined by the injection-molded components from existing within the housing.

[0025] Other aspects of this disclosure are described in the following subsections: (I) electrophoresis apparatus, (II) sample analysis methods, (III) methods for fabricating electrophoresis apparatus, (IV) embodiments, and (V-VI) optional aspects.

[0026] I. Electrophoresis apparatus

[0027] This section outlines an exemplary electrophoresis apparatus with minimal UV-induced autofluorescence; see [link to relevant documentation]. Figure 1-7 .

[0028] Figure 1 and Figure 2An exemplary apparatus 50 ("electrophoresis apparatus") for separating sample components from each other by gel electrophoresis based on size and / or charge is shown. Apparatus 50 may include a housing 52 (interchangeably referred to as a "container" or "receptacle"). Optionally, a plate-like gel 54 having a gel matrix formed of polyacrylamide may be formed within and contained by the housing 52. As the plate-like gel forms in the housing 52, comb teeth 56 may be used to create pores 58 along the top edge of the plate-like gel 54, and the comb teeth 56 may then be removed to allow sample loading into the pores 58. A sealing element 60, such as an adhesive tape strip, may seal the bottom edge of the housing 52 during gel formation to prevent liquid leakage and may then be removed (e.g., by a user) prior to gel electrophoresis. Exemplary sample components that can be separated from each other by electrophoresis include proteins, peptides, nucleic acids, etc.

[0029] The housing 52 may have a front panel 62 and a rear panel 64 that are opposite to and connected to each other. The panels 62 and 64 form an observation window 65, which includes a front window pane 66 and a rear window pane 68, each located between a pair of side portions 70a, 70b or 72a, 72b (see [reference]). Figure 2 and 5 -7). The front panel 62 and the rear panel 64 may be bonded together or otherwise connected to each other at the side portions 70a and 72a and the side portions 70b and 72b, thereby creating a fluid seal along each side of the cavity 74 defined by the housing 52 between the front panel 66 and the rear panel 68 (see...). Figure 5-7 In the described embodiment, housing 52 has only two discrete components (i.e., front and rear panels 62, 64), but in other embodiments, one or more additional components may be included, such as discrete lateral spacers that separate the plates from each other along their sides.

[0030] Cavity 74 is configured to accommodate plate-shaped gel 54. Cavity 74 may be a planar cavity having a pair of opposing planar walls 76, 78, wherein the planar walls 76, 78 are provided by the respective inner surfaces of the front plate 66 and the rear plate 68. Cavity 74 may have a uniform depth that can be measured between the planar walls 76, 78. The sides of cavity 74 may be formed by one or two corresponding side portions 70a, 72a and one or two corresponding side portions 70b, 72b along the respective side regions of the housing 52. During the formation of plate-shaped gel 54, the bottom edge of cavity 74 may be sealed by sealing element 60, and then opened before electrophoresis by removing the sealing element (see also...). Figure 1 Before the formation of the plate-like gel 54, the top edge of the cavity 74 can receive the teeth 80 of the comb 56, and then the comb 56 can be removed before the sample is loaded into the pores 58 of the plate-like gel 54 (see, for example, see...). Figure 1 and 2 ).

[0031] The front panel 62 may have a pair of ears 82a, 82b extending upward from their respective side portions 70a, 70b (see...) Figure 1 and 2 The lugs 82a and 82b together with the front window pane 66 of the front plate 62 form a barrier to retain the running buffer during gel electrophoresis in the electrophoresis apparatus 50.

[0032] The viewing window 65 extends from the outer surface 86 of the front panel 66 to the outer surface 88 of the rear panel 68 throughout the entire housing 52 (see [link]). Figure 2 and 5 -7). The width of the observation window 65 may correspond to the width of the cavity 74 between side portions 70a and 70b and / or between side portions 72a and 72b.

[0033] The observation window 65 is configured to allow the plate-like gel 54 to receive ultraviolet (UV) radiation in situ, that is, to receive UV radiation when the gel is contained within the cavity 74 between the front window pane 66 and the rear window pane 68. Therefore, the observation window 65 is transparent to UV radiation, such as UV radiation in the 320-400 nm (UV-A), 280-320 nm (UV-B), and / or <280 nm (UV-C) range, etc. An observation window that is "UV-transparent" means that most of the UV radiation within a specific wavelength range incident on the observation window passes through the observation window and / or is transmitted to the plate-like gel located in the cavity between the front window pane 66 and the rear window pane 68. For example, the majority of the transmitted UV radiation may be at least 70%, 80%, 85%, or 90%, etc.

[0034] The observation window 65 is also configured to minimize UV-induced autofluorescence. In the absence of the plate-like gel 54, the observation window defines a window of autofluorescence that can be induced by UV irradiation of a specific wavelength. To minimize interference from UV-induced autofluorescence and to allow for in-situ derivatization of proteins to enhance their fluorescence, as well as imaging of the plate-like gel 54, the window autofluorescence per unit area can, for example, be less than 500%, 400%, 300%, 200%, 150%, or 100% of the gel autofluorescence of the plate-like gel 54 under the same unit area and the same UV irradiation. As used herein, autofluorescence refers to background fluorescence produced by one or more substances other than one or more fluorophores of interest. Any fluorescence or autofluorescence disclosed herein may include visible light and / or ultraviolet light, etc.

[0035] Each plate 62, 64 can define its respective polymer injection position 90a, 90b located outside the observation window 65 (see...). Figure 4The polymer injection location of the molded part is the location where the polymer enters the mold that forms the part. The polymer injection location may correspond to the gate location where the polymer enters the mold. If plates 62 and 64 are formed by injecting polymer into a pair of respective molds, for example at polymer injection locations 90a and 90b, the inventors have observed that each polymer injection location 90a and 90b can be locally associated with autofluorescent "scars" that have undesirable high levels of UV-induced autofluorescence (see also Example 1). Therefore, the respective polymer injection locations are placed outside the observation window 65, as... Figure 4 Within one of the shaded areas, this can prevent undesirable high levels of UV-induced autofluorescence from interfering with in-situ imaging of the plate-like gel 54. For example, in the described embodiment, the front plate 62 defines a polymer injection site 90a in one of the side regions 92a or 92b (i.e., in the side portion 70a), while the rear plate 64 defines a polymer injection site 90b at its upper edge portion 94 above the observation window 65. In other embodiments, plates 62 and 64 can each define their respective polymer injection sites, each located in one of the side regions 92a or 92b.

[0036] Figure 6 and Figure 7 A cross-sectional view of the housing 52 is shown without the plate-like gel 54. The front plate 62, rear plate 64, and cavity 74 can have any suitable relative dimensions measured orthogonally to the observation window 65. For example, the front plate 62 and rear plate 64 can each have thicknesses 96 and 98, respectively, which are greater than, equal to, or less than the depth 100 of the cavity 74. Smaller plate thicknesses 96 and 98 can be used to reduce the autofluorescence contribution associated with the thickness of the plates 62 and 64, while still providing sufficient structural strength for the housing 54. A larger cavity depth 100 can be used to increase the sample capacity of the plate-like gel 54, thereby increasing the fluorescence of sample components relative to the observation window 65 of the housing 52.

[0037] Slab gels can be configured to separate proteins in a sample by electrophoresis (e.g., polyacrylamide gel electrophoresis (PAGE)). Therefore, slab gels can be aqueous gels comprising any combination of a gel matrix (e.g., polyacrylamide, agarose, starch, etc.), electrolytes, amphoteric surfactants (e.g., sodium dodecyl sulfate (SDS)) that denature the proteins and ensure that each protein has a substantially equal charge per unit mass (for SDS-PAGE), a reducing agent (e.g., β-mercaptoethanol), and modifiers. Any suitable concentration of the gel matrix can be used for the gel, for example, 5-25% for polyacrylamide gels or 0.5% to 3% for agarose gels. In some embodiments, the modifier can be configured to react with proteins that are driven into the gel by electrophoresis. This reaction can form fluorescent derivatizations of the proteins in response to ultraviolet light irradiation through the observation window. In some embodiments, the modifier can be a haloalkane, such as trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform, or iodoacetic acid.

[0038] Device 50 can be connected to a housing assembly. This housing assembly may have an upper housing and a lower housing to contain respective electrolyte solutions. The upper end of device 50 can be sealed to the upper housing (e.g., via a gasket) such that the respective electrodes are located within their respective housings. A voltage is applied between the respective electrodes to drive the electrophoretic movement of the charged components.

[0039] II. Methods for Sample Analysis

[0040] This section describes exemplary methods for sample analysis using any of the electrophoresis apparatuses disclosed herein (such as in Section 1); see also Figure 8 . Figure 8 The steps described in sample analysis method 110 can be performed in any suitable order and combination.

[0041] As shown in 112, one or more samples can be loaded into one or more wells of a gel housed within a housing. The gel and housing can have any suitable combination of structures and features, such as those described in Section 1 above. Sample loading can be performed before or after the electrophoresis apparatus is connected to the housing assembly (see Section 1), and thus before or after contacting the upper and lower edges of the gel with their respective electrolyte solutions (run buffers), each contained within a corresponding housing of the housing assembly. Each sample can include one or more sample components, such as one or more proteins.

[0042] As shown in 114, one or more sample components in each sample can be driven within a gel by electrophoresis. A voltage can be applied between a pair of electrodes located in the respective chambers of the chamber assembly, thereby generating an electrophoretic potential between the upper and lower edges of the gel. This electrophoretic potential drives the sample components in each sample to enter the gel from their respective wells and move along channels extending from the wells towards the lower edge of the gel. Each sample component can migrate at a rate inversely proportional to its molecular weight. Therefore, electrophoresis can separate sample components according to their size, with larger components moving more slowly and closer to the wells at the end of electrophoresis.

[0043] As shown in 116, sample components in the gel can be derivatized under ultraviolet (UV) irradiation while the gel remains within the housing. In other words, the UV-driven reaction between the modifier and the sample components can occur in situ without opening the housing. The derivatization process of the sample components with the modifier can be performed before, during, and / or after electrophoresis in step 114. In some embodiments, the gel (and / or running buffer) may include a modifier. In some embodiments, the modifier may chemically react with the sample components, and this chemical reaction is driven by UV light. UV light can penetrate at least a portion of the housing containing the gel to reach the gel, for example, through at least one layer of window panes in the housing's viewing window, thereby irradiating the gel and the substances therein (e.g., proteins and modifiers) with UV light. The UV light can have any suitable wavelength, such as 320-400 nm (UV-A), 280-320 nm (UV-B), and / or less than 280 nm (UV-C), etc. The irradiation can be performed (i) after the sample is loaded into the well and before electrophoresis begins, (ii) during electrophoresis of the sample components in the gel, and / or (iii) after the electrophoretic potential has been removed but while the gel is still between the panes of the observation window. In some embodiments, the irradiation can promote the formation of fluorescent derivatizations of at least one protein in each sample. In some embodiments, the modifying agent for protein derivatization can be a haloalkane, such as trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform, or iodoacetic acid. In some embodiments, the modifying agent can chemically react with amino acid residues present in the protein, such as tryptophan residues.

[0044] As shown in 118, fluorescence can be induced in the gel while it remains within the housing. In other words, the derivatized fluorescent sample components in the gel can be excited in situ without opening the housing. Fluorescence can be induced by irradiating the gel with ultraviolet light through at least one pane of the viewing window of the housing. The gel can be irradiated through the front pane, rear pane, or both panes of the viewing window. The ultraviolet light can be of any suitable wavelength, such as 320-400 nm (UV-A), 280-320 nm (UV-B), and / or less than 280 nm (UV-C), etc. In some embodiments, ultraviolet light of the same wavelength can be used for steps 116 and 118 of method 110, thereby allowing steps 116 and 118 to be performed in parallel.

[0045] As shown in 120, the fluorescence induced in step 118 can be detected. Detecting fluorescence may include capturing an image formed by the fluorescence. Alternatively or additionally, detecting fluorescence may also include monitoring fluorescence to determine when the reaction of the modifier with the sample component (e.g., a fluorescence threshold) reaches predetermined conditions. Monitoring fluorescence can determine when the derivatization reaction and / or imaging are sufficiently completed and thus stopped.

[0046] III. Methods for fabricating electrophoresis apparatus

[0047] This section describes an exemplary method for fabricating an electrophoresis apparatus with low autofluorescence, thereby enabling in-situ treatment of the gel contained within the housing of such apparatus (e.g., in Section 1); see also Figure 9 . Figure 9 The steps described in Method 130 for making an electrophoresis apparatus can be performed in any suitable order and combination.

[0048] As shown in 132, multiple discrete parts can be injection molded, each defining a location for polymer injection. In some embodiments, a pair of flat plates can be injection molded. This pair of flat plates can be configured as window panes providing an observation window. During the formation of the discrete parts, the polymer injection location of each discrete part can be connected to but spaced apart from the corresponding window pane. In some embodiments, the polymer injection location can be removed from the discrete parts after their formation, for example, by cutting or severing it from the body of the discrete parts to at least partially form one of the flat plates.

[0049] The location of polymer injection can exhibit localized autofluorescence relative to other parts of the corresponding component. The level of this localized autofluorescence can be influenced by molding parameters such as heat and pressure. For example, longer heating times and / or higher temperatures (e.g., subjecting the polymer to greater pressure and increased strain) can produce greater autofluorescence. Therefore, to reduce or eliminate locally increased autofluorescence at the polymer injection site, the polymer can be rapidly heated, immediately injected into the mold, and then rapidly cooled to minimize heat-related autofluorescence. Alternatively or supplementarily, the polymer can be injected at a slower rate under lower pressure to reduce pressure / strain-related autofluorescence.

[0050] As shown in 134, these components can be interconnected to form a housing with an observation window that bypasses each polymer injection location of the multiple discrete components. The observation window may include a front window pane and a rear window pane with a cavity between them.

[0051] As shown in 136, a plate-like gel can be formed within the cavity of the housing. The plate-like gel can be formed by gelation using a polymer (such as agarose) or a polymer precursor (such as acrylamide). Because the observation window avoids the location of each polymer injection, the plate-like gel can be imaged after electrophoresis and labeling without imaging any localized autofluorescence sites of the polymer injection into the housing.

[0052] IV. Examples

[0053] This section describes other aspects of the electrophoresis apparatus of this disclosure. These aspects are set forth herein for illustrative purposes and are not intended to limit the scope of this disclosure.

[0054] Example 1. Electrophoresis apparatus containing different polymers

[0055] This embodiment shows images of two embodiments 250 and 250x of the electrophoresis apparatus 50, taken after gel electrophoresis and in-situ derivatization of sample 251 in the plate-like gel 254 of each apparatus 250 and 250x; see also Figure 10 and 11 (See also) Figure 1-7 ).

[0056] Electrophoresis apparatuses 250 and 250x each include housings 252 and 252x for plate-like gels 254. Housing 252 provides an observation window 265 suitable for in-situ derivatization and imaging of the plate-like gels 254 (see [link to relevant documentation]). Figure 10The housing 252 (and viewing window 265) is formed of a polyacrylate polymer (poly(methyl methacrylate)-PMMA), which is UV-transparent and exhibits low UV-induced autofluorescence. More specifically, housing 252 comprises a CA-41UVT-LL2 polymer, which is available from Plaskolite (Columbus, Ohio, USA). In contrast, housing 252x provides a viewing window 265x that does not allow for in-situ derivatization and imaging (see...). Figure 11 The housing 252x (and viewing window 265x) comprises a copolymer of styrene and acrylonitrile (SAN polymer), which is not UV transparent.

[0057] Protein-containing samples 251 are loaded into the wells 258 of each plate-like gel 254. Proteins 267 of the samples 251 are electrophoresed in channels 269 of the separation portions 271 of each plate-like gel 254 by applying an electrophoretic potential. Each observation window 265, 265x is then irradiated with ultraviolet light to promote in-situ derivatization of protein 267 within the plate-like gel 254. This derivatization is achieved by chemically reacting protein 267 with a haloalkane modifier (2,2,2-trichloroethanol) present in the plate-like gel (i.e., incorporated during gel formation). The haloalkane modifier reacts with tryptophan residues of protein 267, altering its fluorescence to emit in the visible spectrum.

[0058] The specific polymer forming the observation windows 265 or 265x determines whether protein 267 in each plate-like gel can be detected in the captured image. To facilitate in-situ derivatization of protein 267, a UV-transparent polymer is required to allow effective UV irradiation of the plate-like gel 254 through at least one pane of the housing 252 or 252x. Therefore, the UV-opaque observation window 265x prevents adequate derivatization of protein 267 with haloalkane labeling. Furthermore, even if protein 267 is derivatized within the observation window 265x, the window does not allow UV excitation of the derivatized protein. Additionally, to detect the fluorescence of the in-situ derivatized protein 267, which can produce a relatively weak fluorescence signal, the autofluorescence of the plate-like gel 254 and the observation window 265 must be very low to produce a sufficient signal-to-noise ratio in the captured image. The polyacrylate used to form the observation window 265 is both UV-transparent and exhibits very little autofluorescence, making it easy to detect the derivatized protein 267 against the background.

[0059] Figure 10 and 11 The images also indicate the respective polymer injection locations 290 and 290x. Figure 10In the image, the polymer injection site 290 in the observation window 265 produces a strong local fluorescence signal because the observation window 265 is ultraviolet transparent and exhibits very little autofluorescence. As described in Section 1 above, moving the polymer injection site 290 outside the observation window 265 advantageously prevents the fluorescence of the polymer injection site 290 from overlapping with that of the protein 267 in the captured image. Figure 11 The polymer injection site 290x is almost invisible through the observation window 265x because the observation window 265x is opaque to ultraviolet light.

[0060] Example 2. Comparison of polyacrylates

[0061] This example describes a comparison of autofluorescence detected in a group of seven commercially available polyacrylate formulations from Plaskolite (Columbus, Ohio, USA); see [link to relevant documentation]. Figure 12 and 13 .

[0062] Figure 12 Fluorescence images of seven different acrylate (AG) samples taken under UV irradiation to induce autofluorescence are shown. Each sample is substantially transparent to the UV light used for irradiation, but the autofluorescence response of the samples to UV irradiation varies considerably. Strong autofluorescence makes the samples appear darker in the images (compared to the background between the samples).

[0063] Figure 13 Showing a list Figure 12 A table showing the relative levels of autofluorescence in seven different polyacrylate samples. Sample A Sample A exhibited the lowest autofluorescence, while sample F (CA-83UVT(N)) showed the highest autofluorescence, exceeding that of sample A by more than 75 times. Identifying and utilizing polymers that are both UV-transparent and exhibit minimal autofluorescence is crucial for the derivatization and imaging of proteins in plate gels through the viewing window of the enclosure containing the plate-like gel.

[0064] V. Aspects of Choice 1

[0065] This section describes aspects of the selection of electrophoresis apparatus and methods of this disclosure as a series of indexed paragraphs.

[0066] Paragraph A1. An electrophoresis apparatus for sample analysis, comprising: (i) a housing defining a cavity between a first pane and a second pane of an observation window; and (ii) a plate-like gel located within the cavity; wherein the observation window is transparent to ultraviolet light, wherein, in the absence of the plate-like gel, the observation window defines window autofluorescence induced by ultraviolet light irradiation, and wherein, under the same unit area and the same ultraviolet light irradiation, the window autofluorescence per unit area is less than 5 times that of the gel autofluorescence of the plate-like gel.

[0067] Paragraph A2. The electrophoresis apparatus as described in paragraph A1, wherein the housing includes a first plate and a second plate, wherein the first plate includes a first window pane and the second plate includes a second window pane, and wherein each plate is (a) an injection-molded part defining a polymer injection location spaced apart from an observation window within the housing, or (b) formed by the injection-molded part at least in part by physically removing the polymer injection location from the injection-molded part.

[0068] Paragraph A3. An electrophoresis apparatus as described in paragraph A2, wherein the housing has locally increased autofluorescence around at least one polymer injection site.

[0069] Paragraph A4. An electrophoresis apparatus as described in paragraphs A2 or A3, wherein at least one of the first plate and the second plate defines a polymer injection location formed by a side portion of the housing or by a top or bottom portion of the first plate or the second plate that does not overlap with the other plate of the housing.

[0070] Paragraph A5. An electrophoresis apparatus as described in any one of paragraphs A1 to A4, wherein the plate-like gel defines a plurality of pores and includes a separation portion having a plurality of channels aligned with the plurality of pores, and wherein the separation portion is contained in an observation window.

[0071] Paragraph A6. An electrophoresis apparatus as described in any one of paragraphs A1 to A5, wherein the slab gel is configured to separate proteins of a sample by electrophoresis, and wherein the slab gel includes a modifier configured to derivatize the proteins to alter their fluorescence.

[0072] Paragraph A7. An electrophoresis apparatus as described in paragraph A6, wherein the modifier is configured to derivatize proteins in the gel in response to ultraviolet light irradiation of the observation window.

[0073] Paragraph A8. The electrophoresis apparatus as described in paragraph A7, wherein the modifier is a haloalkane selected from trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform and iodoacetic acid.

[0074] Paragraph A9. The electrophoresis apparatus as described in any one of paragraphs A1 to A8, wherein the window autofluorescence is less than the gel autofluorescence.

[0075] Paragraph A10. The electrophoresis apparatus as described in any one of paragraphs A1 to A9, wherein the housing is formed of polyacrylate.

[0076] Paragraph A11. The electrophoresis apparatus as described in any one of paragraphs A1 to A10, wherein the window autofluorescence is less than the gel autofluorescence or less than four, three, or two times the gel autofluorescence.

[0077] Paragraph B1. A method for analyzing a sample, the method comprising: (i) electrophoresis of one or more proteins of a sample contained in a plate-like gel in a housing, the housing comprising a first plate, a second plate, and an observation window, the observation window being transparent to ultraviolet light and extending through the housing from the outer surface of the first plate through the plate-like gel to the outer surface of the second plate; (ii) irradiating at least a portion of the observation window of the housing with ultraviolet light while the plate-like gel is contained in the housing; and (iii) detecting fluorescence of one or more proteins or derivatives thereof, the fluorescence being induced by irradiation and detected after propagation through at least one of the first and second plates of the observation window; wherein, in the absence of the plate-like gel, the observation window has an autofluorescence per unit area in response to irradiation that is less than five times the background fluorescence of the plate-like gel.

[0078] Paragraph B2. The method as described in paragraph B1, wherein irradiation causes a modifier in the gel to chemically react with one or more proteins in the sample to form a derivatization of said one or more proteins, wherein detecting fluorescence includes detecting fluorescence emitted by the derivatization.

[0079] Paragraph B3. The method as described in paragraph B2, wherein the modifier is a haloalkane selected from trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform and iodoacetic acid.

[0080] Paragraph B4. The method as described in any one of paragraphs B1 to B3, wherein detecting fluorescence includes capturing one or more images formed by fluorescence.

[0081] Paragraph B5. The method as described in paragraph B4, wherein capturing stops when the detected fluorescence reaches one or more predetermined criteria.

[0082] Paragraph B6. The method as described in any one of paragraphs B2 to B5, wherein detecting fluorescence includes monitoring fluorescence to determine when the reaction of the modifier reaches predetermined conditions, and wherein, optionally, irradiation is stopped when the predetermined conditions are reached.

[0083] Paragraph B7. An electrophoresis apparatus as described in any one of paragraphs B1 to B6, wherein each plate is (a) an injection-molded part defining a polymer injection position spaced apart from an observation window, or (b) formed by the injection-molded part at least in part by physically removing the polymer injection position from the injection-molded part.

[0084] Paragraph B8. The method as described in any one of paragraphs B1 to B7, wherein the window autofluorescence is less than the background autofluorescence or less than four, three, or two times the background autofluorescence.

[0085] Paragraph C1. A method of forming an electrophoresis apparatus for sample analysis, the method comprising: (i) injection molding two or more discrete components, wherein each discrete component defines a polymer injection site; (ii) interconnecting at least a portion of each of the two or more discrete components to form a housing, the housing defining a cavity between a first pane and a second pane of a double-layered observation window, wherein each polymer injection site (a) is included in the housing and avoids the observation window or (b) is not in the housing; and (iii) forming a plate-like gel in the cavity; wherein the observation window is transparent to ultraviolet light, wherein, in the absence of the plate-like gel, the observation window has window autofluorescence induced by ultraviolet light irradiation, and wherein, under the same unit area and the same ultraviolet light irradiation, the window fluorescence per unit area is less than 5 times the gel autofluorescence of the plate-like cell.

[0086] Paragraph C2. The electrophoresis apparatus as described in paragraph C1, wherein the housing has locally increased autofluorescence around at least one polymer injection site.

[0087] Paragraph C3. The method as described in paragraphs C1 or C2, wherein the first pane is formed of a first plate, wherein the second pane is formed of a second plate, wherein at least one of the first and second plates defines a polymer injection location formed by a side region of the housing or by a top or bottom portion of the first or second plate that does not overlap with the other plate.

[0088] Paragraph C4. The method as described in any one of paragraphs C1 to C3, wherein the first pane is provided by a plate, further comprising physically removing a polymer injection site from one of the discrete components to at least partially form the first plate, wherein, optionally, the removal comprises cutting or breaking the discrete component to remove the polymer injection site.

[0089] Paragraph C5. The method as described in any one of paragraphs C1 to C3, wherein the connection comprises bonding a pair of two or more discrete components together.

[0090] Paragraph C6. The method as described in any one of paragraphs C1 to C5, wherein the window autofluorescence is less than the gel autofluorescence or less than four, three, or two times the gel autofluorescence.

[0091] As used in this disclosure, the term “exemplary” means “illustrative” or “used as an example” and does not imply preference or superiority.

[0092] The foregoing disclosure may include several different inventions with independent utility. While various of these inventions have been disclosed in their preferred forms, the specific embodiments of the invention disclosed and illustrated herein should not be considered limiting, as many variations are possible. The subject matter of this invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or characteristics disclosed herein. Certain novel and non-obvious combinations and sub-combinations are specifically pointed out in the following paragraphs. Inventions embodied in other combinations and sub-combinations of features, functions, elements, and / or characteristics may be claimed in applications claiming priority to this application or related applications. Such paragraphs, whether referring to different or identical inventions, and whether broader, narrower, identical, or different from the scope of the original paragraph, are also considered to be included within the subject matter of the invention disclosed herein. Furthermore, ordinal designations used to identify elements, such as first, second, or third, are used to distinguish elements and do not indicate a specific position or order of these elements unless otherwise specifically stated.

Claims

1. An electrophoresis apparatus for sample analysis, comprising: A cavity is defined between the first and second panes of the observation window; as well as A plate-like gel located within a cavity; The observation window is transparent to ultraviolet light, and in the absence of a plate-like gel, the observation window defines window autofluorescence that can be induced by ultraviolet light irradiation, and the window autofluorescence per unit area is less than five times that of the gel autofluorescence of the plate-like cell under the same unit area and the same ultraviolet light irradiation. The housing includes a first plate and a second plate, wherein the first plate includes a first window pane and the second plate includes a second window pane, and wherein each plate is (i) an injection-molded part defining a polymer injection location spaced apart from an observation window, or (ii) formed by the injection-molded part at least in part by physically removing the polymer injection location from the injection-molded part; The polymer injection site corresponds to an autofluorescent scar.

2. The electrophoresis apparatus as described in claim 1, wherein, The housing has locally increased autofluorescence around at least one polymer injection site.

3. The electrophoresis apparatus as described in claim 1, wherein, At least one of the first plate and the second plate defines a polymer injection location formed by a side portion of the housing or by a top or bottom portion of the first plate or the second plate that does not overlap with the other plate.

4. The electrophoresis apparatus as described in claim 1, wherein, The plate-like gel defines a plurality of pores and includes a separation portion having a plurality of channels aligned with the plurality of pores, wherein the separation portion is contained within an observation window.

5. The electrophoresis apparatus as claimed in claim 1, wherein, The plate-like gel is configured to separate proteins from a sample by electrophoresis, and wherein the plate-like gel includes a modifier configured to derivatize the protein to alter its fluorescence.

6. The electrophoresis apparatus as described in claim 5, wherein, The modifier is configured to derivatize proteins in the gel in response to ultraviolet light irradiation of the observation window.

7. The electrophoresis apparatus as claimed in claim 6, wherein, The modifier is a haloalkane selected from trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform, and iodoacetic acid.

8. The electrophoresis apparatus of claim 1, wherein the window autofluorescence is less than the gel autofluorescence.

9. The electrophoresis apparatus of claim 1, wherein the housing is formed of polyacrylate.

10. A method for analyzing a sample, the method comprising: Electrophoresis is performed on one or more proteins in a plate-like gel contained in a container, the container including a first plate, a second plate, and an observation window, the observation window being transparent to ultraviolet light and extending from the outer surface of the first plate through the plate-like gel to the outer surface of the second plate and through the container. While the plate-like gel is contained within a housing between a first plate and a second plate, at least a portion of the observation window of the housing is irradiated with ultraviolet light; and Detecting the fluorescence of one or more proteins or their derivatives, wherein the fluorescence is induced by irradiation and detected after propagation through at least one of the first and second plates of the observation window; In the absence of a plate-like gel, the observation window exhibits an autofluorescence per unit area of ​​less than five times that of the background fluorescence of the plate-like gel. Each plate is (i) an injection-molded part that defines a polymer injection location spaced apart from an observation window, or (ii) formed by the injection-molded part at least in part by physically removing the polymer injection location from the injection-molded part; The polymer injection site corresponds to an autofluorescent scar.

11. The method of claim 10, wherein irradiation causes a modifier in the gel to chemically react with one or more proteins in the sample to form a derivatization of the one or more proteins, wherein detecting fluorescence includes detecting fluorescence emitted by the derivatization.

12. The method of claim 11, wherein the modifier is a haloalkane selected from trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform and iodoacetic acid.

13. The method of claim 10, wherein detecting fluorescence comprises capturing one or more images formed by fluorescence.

14. The method of claim 13, wherein the capture stops when the detected fluorescence reaches one or more predetermined criteria.

15. A method for forming an electrophoresis apparatus for sample analysis, the method comprising: Injection molding of two or more discrete parts, wherein each discrete part defines a polymer injection location; At least a portion of each of two or more discrete components is interconnected to form a housing that defines a cavity between a first pane and a second pane of a double-viewing window, wherein each polymer injection location is (i) included in the housing and bypasses the viewing window or (ii) is not in the housing; as well as A plate-like gel forms within the cavity; The observation window is transparent to ultraviolet light, and in the absence of a plate-like gel, the observation window has window autofluorescence that can be induced by ultraviolet light irradiation, and the window fluorescence per unit area is less than five times that of the gel autofluorescence of the plate-like cell under the same unit area and the same ultraviolet light irradiation. The polymer injection site corresponds to an autofluorescent scar.

16. The method of claim 15, wherein the housing has locally increased autofluorescence around at least one polymer injection site.

17. The method of claim 15, wherein the first pane is formed of a first plate, wherein the second pane is formed of a second plate, wherein at least one of the first and second plates defines a polymer injection location formed by a side region of the housing or by a top or bottom portion of the first or second plate that does not overlap with the other plate.

18. The method of claim 15, wherein, The first pane is provided by a plate, which further includes physically removing a polymer injection site from one of the discrete components to at least partially form the first plate.

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