Carbon Black Nanoparticles in Non-Instrumental Immunoassays Development for Diagnostic Applications

Abstract

Due to their unique physicochemical properties, carbon black nanoparticles represent a promising alternative for solving analytical problems. However, diagnostic reagents based on carbon black nanoparticles have not yet found widespread practical application. This review examines the development and application of carbon black nanoparticle conjugates with recognition molecules as diagnostic reagents in test systems that enable non-instrumental interpretation of results. The review critically evaluates the methods for synthesis and characterization of carbon black-based diagnostic reagents. Furthermore, the review summarizes and discusses existing studies comparing the effectiveness of carbon black nanoparticle-based bioconjugates with traditional colorimetric labels. The scientific articles included in the review were carefully analyzed for the presence of an assessment of the reproducibility of methods for obtaining diagnostic reagents based on carbon black nanoparticles and their long-term storage. The main challenges and future prospects of using carbon black nanoparticles in immunoassays are discussed.

1. Introduction

The 2020 pandemic highlighted that developing and improving diagnostic methods remains an important and unresolved challenge. Among the widely accessible, simple diagnostic techniques for rapid, non-instrumental assessment, immunochromatographic analysis has become the gold standard [1,2].

Tests of this type must meet several key criteria: simplicity, storage stability, and suitability for mass production [1]. Immunochromatographic tests demonstrate high sensitivity and specificity, often reaching 97–99% [3]. Nevertheless, developing methods to improve the performance of rapid tests remains important. Using such tests involves addressing challenges such as ensuring long-term reagent stability, reducing analysis time, preventing non-specific sorption, and increasing the signal-to-noise ratio [4,5,6].

The search for solutions to these problems will lead to the development of new approaches to the design of more universal, reliable, and convenient rapid diagnostic methods suitable for both the scientific community and medical personnel, as well as non-professional users [5,7].

Traditional immunochromatographic assays employ color labels, including colloidal gold nanoparticles, latex particles, and fluorescent nanoparticles, which produce an optical signal that can be visually detected [2]. However, these labels are ineffective when the target ligand is present at ultra-low concentrations due to their low molar absorption coefficient, necessitating a large number of nanoparticles to generate a detectable optical signal [8,9]. Therefore, one key direction for improving analytical methods is to explore new approaches for synthesizing diagnostic reagents based on more effective color labels.

Since the 1990s, information has been emerging on the possibility of using carbon black nanoparticles as colorimetric labels in the design of analytical systems for visual assessment [10]. Conjugates based on carbon nanoparticles have a higher absorption coefficient and are intensely colored, which provides a high level of analytical signal and a high signal-to-noise ratio, allowing us to significantly reduce the detection limit of the analysis.

Carbon nanoparticles have a larger surface area compared to gold nanoparticles, which makes their functionalization with recognition molecules more efficient.

Moreover, this is a widely available material with standardized properties and production process, which is important when introducing nanomaterials into real clinical practice. It has been demonstrated that the use of carbon black nanoparticles as a color label in immunochromatographic analysis allows to reduce the detection limit several times compared to gold nanoparticles [11,12].

This review explores carbon black nanoparticle–based test systems that enable visual result assessment (e.g., color change or line appearance) without instrumentation. Their significance arises from the urgent need for rapid, affordable, and accessible diagnostics in resource-limited settings, such as remote locations or field first-aid scenarios. Such technologies underline modern rapid tests, including those for COVID-19, confirming their continued relevance. The current task is to review and critically analyze information on the use of CBNPs in the context of creating analytical systems with non-instrumental visual assessment of results, as well as identify new promising areas of research in the field of developing such methods. The novelty of this review lies in a thorough examination of the existing scientific studies focused on the development of immunoassays based on CBPNs bioconjugates, particularly in terms of evaluating the reproducibility of the methods used to prepare these conjugates and their long-term storage.

A literature search was conducted using the PubMed and ScienceDirect databases. Articles published up to and including 2025 were considered, applying the following keywords: carbon black nanoparticles, carbon black, immunoassay, non-instrumental immunoassay, and rapid immunoassay. This review focused exclusively on research articles describing the development of immunoassays aimed at non-instrumental outcome assessment. Studies involving immunoassays based on allotropic forms of carbon other than carbon black nanoparticles were excluded. Applying these criteria, a total of forty relevant articles were identified for inclusion.

2. Carbon Black Nanoparticles, General Information

2.1. Characteristics of Carbon Black Nanoparticles

The term carbon black nanoparticles (CBNPs) in the literature usually refers to derivatives of carbon black (CB) or soot. Although the terms «carbon black» and «soot» are often used interchangeably, these materials differ slightly in their physicochemical properties. Soot is a by-product of incomplete combustion of carbonaceous materials and is a powdery mass of small black particles [13]. Examples of soot include coal, charred wood, petroleum coke, cenospheres, and resins. CBNPs can also be produced from soot by burning a candle in the laboratory [14,15,16].

Carbon black (CB) is produced under controlled conditions for commercial use in the rubber, printing, and paint industries [17,18,19]. It is formed by the partial combustion of heavy petroleum materials such as coal tar, ethylene cracking tar, and catalytic cracking tar [20,21].

CB and soot are forms of amorphous carbon (a-C). It is described as free, chemically active carbon that has no crystalline structure, which distinguishes it fundamentally from other allotropes of carbon. According to IUPAC, it is a carbon material without long-range or medium-range crystalline order. Short-range order exists, but with variations in interatomic distances and/or bond angles. The atoms in amorphous carbon are sp3 hybridized. The structure may include aromatic rings, with the rings arranged at different angles to each other and may form subclusters within the particle, but the “pattern” of the composition of the rings and non-ring atoms is not repeated, that is, there is no rule for their arrangement. Since the atoms of aromatic rings have sp2 hybridization, amorphous carbon materials can be characterized by the ratio of the number of sp3 hybridizations to the number of sp2 hybridizations, or, in other words, aromatization [22,23,24,25]. Unlike aromatic rings, in which electrons are delocalized, in an amorphous structure electrons are localized, which leads to the possibility of various local processes (addition of atoms, dissociation) [26,27].

The presented features determine the unique properties and versatility of amorphous carbon, and, accordingly, the properties of carbon black nanoparticles based on soot and carbon black [24]. Structural differences of carbon nanoparticles from other nanomaterials lie in their formation and assembly. Graphene nanoparticles exist in the form of flat two-dimensional sheets, and carbon nanotubes form seamless cylindrical tubes, carbon nanoparticles exist in the form of fused spherical primary particles that combine into complex three-dimensional chain or grape-like structures with sizes from 10 to 1000 nm. Such aggregation behavior and amorphous nature lead to the fact that carbon black has a higher surface area to volume ratio.

Carbon black and soot are obtained by different methods and may have minor differences in physicochemical properties resulting from their production process. However, in the analyzed articles, nanoparticles obtained from both CB and soot are referred to as “carbon black nanoparticles” and are considered nanoparticles of amorphous carbon. Therefore, in the rest of our manuscript, we use a single term for both CB and soot.

2.2. CB Preparation Methods

The process of producing carbon black occurs through a dehydrogenation reaction to form atomic carbon or C2 radicals, which then condense into solid carbon.

CB production methods are divided into two main ones based on the type of reaction occurring during the production process: thermal oxidation and thermal decomposition [28,29].

Thermal oxidation includes four main methods: the production of blast furnace black, gas black, lamp black and channel black. The production of CB began with the Channel Black process, founded in the USA and using natural gas as a raw material. The process is based on the contact of cold surfaces with a flame. This is almost obsolete technology due to low productivity and environmental issues [30,31].

The German company Degussa pioneered a method for obtaining CB from coal tar —Gas Black process. This is a more environmentally friendly and safe technology [31]. Gas Black process (Degussa method) involves the use of distillates or oils of coal tar, which are evaporated and transported by a high-hydrogen-containing gas into a tubular chamber with many burners. The resulting flame interacts with water-cooled drums. Some of the CB settles on the drums, and the rest is filtered. Primary particles have a size of 10 to 30 nm, and aggregates are characterized by a loose structure and high dispersion. The surface of CB particles undergoes oxidation when exposed to high temperatures in the presence of oxygen, resulting in the formation of acidic oxides. Consequently, when suspended in water, CB exhibits an acidic reaction, with a pH range of 4 to 6. To enhance the acidity of the surface further, oxidative post-treatment is commonly employed using substances such as nitrogen dioxide or ozone, which significantly increases the number of acidic surface groups. In such cases, the oxygen content can reach as high as 15% or more. This method is used to obtain carbon blacks with a high structure and a large surface, such as Special Black 4 (Spezial Schwartz 4) [30,31]. Thus, in the 1990s, van Amerongen proposed using technical carbon Spezial Schwartz 4 to create bioconjugates of CBNPs with recognition molecules with their subsequent use as diagnostic reagents [10].

Lamp black is obtained in vacuum by heating aromatic oils in a cast iron boiler under a refractory hood, which causes partial combustion with condensation of CB vapors. The product is captured by filtration after the cooling stage. This method results in reduced oxidation and fewer acidic functional groups due to lower oxygen content in the reaction, despite the occurrence of partial combustion. Modern lamp black factories produce standardized grades of Specialty and Rubber Carbon Blacks with a wide variation in the size of primary particles (from 60 to more than 200 nm), which, combined with a neutral-alkaline reaction (a pH range of 6 to 9), prevents their use in the synthesis of diagnostic reagents [28,32].

Today, the most common industrial method for obtaining CB, which accounts for more than 95% of production, is the Furnace Black process. The process uses liquid hydrocarbons as feedstock, with natural gas providing heat [28]. The feedstock is atomized and fed into a high-temperature, refractory-lined furnace where pyrolysis occurs. The resulting soot is immediately cooled with water to prevent side reactions. The gas stream was cleaned in a heat exchanger and filtration system, after which the soot is collected and granulated with water and binders or compacted to facilitate subsequent processing and incorporation into polymers. The aqueous suspension exhibits a pH range of 6 to 10, which is attributed to the presence of small quantities of basic oxides on the particle surface. Notably, the Furnace Black method provides the greatest degree of control among all production methods, enabling the degree of aggregation and structure to be manipulated through the addition of alkali metal salts. Special types of carbon black, such as oil-extended carbon black, are developed to enhance dispersibility and reduce dustiness. The primary particle size ranges from 10 to 80 nm and the aggregates determine the structure and functional properties of the material [28,33,34]. An example is Special Black 100 carbon black with small primary particles and a high specific surface area, which has improved brightness properties. This type of carbon black has been successfully used to develop diagnostic reagents for immunochromatographic test systems [12,35].

Thermal decomposition processes include the Thermal Black Process and the Acetylene Black Process. Thermal Black is based on the cyclic operation of two furnaces: one is preheated, and the other undergoes oxygen-free thermal decomposition of natural gas. This process yields carbon black particles with sizes ranging from 300 to 500 nm for medium decomposition, or 120 to 200 nm for fine decomposition. The latter option is less commonly employed. The properties of carbon black produced via this method differ significantly from those obtained through thermal oxidation methods, primarily due to the absence of oxygen during formation, which results in a pH range of 7 to 9. Furthermore, this CB exhibits low j low pigmentation and a smaller specific surface area, characteristics that are attributed to the size of its primary particles. Consequently, it is not suitable for the synthesis of diagnostic reagents [28,29,32,33,34].

Acetylene black is obtained by high-temperature exothermic decomposition of acetylene, often with the addition of hydrocarbons to regulate the reactor temperature. The final product is a high-purity, highly conductive carbon black with particles measuring 32–42 nm. CB obtained by this method is characterized by an alkaline or neutral reaction, with a pH value ranging from 5 to 8. Acetylene black is characterized by a very high structure. Although the median size of primary particles is comparable to Gas Black and Furnace Black, its structure differs significantly from the spherical shape, which determines the use of acetylene carbon black in polymers and batteries [28,33,34].

The use of plasma technologies in the production of carbon black is a relatively new direction that allows you to create materials with unique properties. This method has environmental benefits and increased efficiency, providing 100% carbon black output from the original raw material and obtaining pure hydrogen as a by-product. However, an economically attractive industrial plasma process has not yet been created [33,34,36,37,38,39].

Thus, the considered physicochemical and technological features of carbon black and its nanoparticles ensure its wide use in various industries in the creation of highly effective materials and analytical methods.

3. Detection Reagents Based on Carbon Black Nanoparticles

3.1. Functionalization of Carbon Black Nanoparticles with Recognition Molecules

As noted earlier, CBNPs have significant potential for biomodification due to their high specific surface area and the variety of available grades, which allows selecting the optimal type to suit specific biomodification purposes and methods [28,32]. The main approach to conjugating CBNPs s with recognition molecules (Table 1) is the physical adsorption of protein molecules such as immunoglobulins, neutravidin, and streptavidin onto nanoparticles surface. Therefore, it is important to study the mechanisms underlying the interaction of CB nanoparticles with proteins. In the work [40] the interaction of CBNPs with the inert protein bovine serum albumin was studied. The authors showed that treatment with the protein increases the stability of the nanoparticles (the zeta potential decreases from −3.9–2.7 to −24.3 and −23.6 after the addition of BSA to the particles, the hydrodynamic diameter of the particles decreased by 17–64 nm after incubation with BSA). Using SEM images, it was demonstrated that the interaction of CBNP with the protein does not lead to significant changes in their morphology. However, the ternary diagram analyzed using SEM-EDX suggested that BSA was bound to the particle surface, resulting in a shift in the ratios of the nanoparticles to the ratios of BSA [40].

As can be seen from Table 1, most of the CBNPs used in the development of tests are derivatives of carbon black SB4 (Special Black 4) from Degussa AG. Also, nanoparticles based on this brand were first used as a colorimetric label in immunoassay for visual reading of results [10,41,42]

Table 1. Characteristics of CBNP-based detection reagents.

No	CBNP’s Sources	Bioconjugation Method	Recognition Molecule	Ref.
1	Spezial Schwartz 4	physical adsorption	anti-albumin antibody	[10]
2	Spezial Schwartz 4	physical adsorption	anti-alpha-amylase antibody	[43]
3	Spezial Schwartz 4	physical adsorption	monoclonal antibody	[44]
4	N/S	physical adsorption	Mouse anti-rat IgG F(ab′)2 fragment specific antibody	[45]
5	Spezial Schwartz 4	physical adsorption	Anti-IgE immunoglobulin G	[46]
6	N/S	physical adsorption	monoclonal antibodies vs Verotoxin-Producing Escherichia coli	[47]
7	Carbon nanoparticles, in the form of nanostrings, were purchased from Maiia Diagnostics (Uppsala, Sweden)	physical adsorption	anti-influenza A nucleoprotein Monoclonal antibody	[48]
8	carbon black anti-EPO suspension, MAIIA Diagnostics	physical adsorption	anti-EPO antibody	[49]
9	N/S	N/S	anti-SFTSV antibody	[50]
10	Carbon black N115	physical adsorption	Mouse anti-human IgG monoclonal antibody	[51]
11	Carbon black 100	physical adsorption	Dengue Virus NS1 glycoprotein mouse monoclonal antibody	[12]
12	Spezial Schwartz 4	physical adsorption	avidin	[11]
13	Spezial Schwartz 4	physical adsorption	antibody	[52]
14	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[4]
15	N/S	physical adsorption	neutravidin	[53]
16	Spezial Schwartz 4	physical adsorption	neutravidin	[54]
17	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[55]
18	Candle soot	physical adsorption followed by treatment with glutaraldehyde	monoclonal antibody and receptor	[14]
19	Candle soot	physical adsorption followed by treatment with glutaraldehyde	monoclonal antibody	[15]
20	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[56]
21	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[57]
22	Spezial Schwartz 4	physical adsorption	neutravidin	[58]
23	Special blacks and other grades from Degussa AG	physical adsorption	monoclonal antibody	[59]
24	Spezial Schwartz 4	physical adsorption	scCro DNA binding protein	[60]
25	Maiia Diagnostics	physical adsorption	antibiotin antibody or Oligonucleotide	[61]
26	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[62]
27	Special Black 100	covalent cross-linking by a Silane	monoclonal antibody	[35]
28	Spezial Schwartz 4	physical adsorption	neutravidin	[63]
29	Amorphous CNPs	physical adsorption	Polyclonal antibody	[64]
30	Spezial Schwartz 4	physical adsorption	p48 protein	[65]
31	Spezial Schwartz 4	physical adsorption	neutravidin	[66]
32	Spezial Schwartz 4	physical adsorption	Goat anti-Mouse IgG FcY	[67]
33	Candle soot	covalent cross-linking with glutaraldehyde	monoclonal antibody	[16]
34	Degussa, Düsseldorf, Germany	physical adsorption	polyclonal antibodies	[68]
35	N/S	physical adsorption	monoclonal antibody	[69]
36	Special Black 4	physical adsorption	monoclonal antibody	[70]
37	Candle soot	covalent cross-linking with glutaraldehyde and biotin-streptavidin interaction	DNA-aptemer And biotin	[71]
38	Special Black 100	physical adsorption	monoclonal antibody	[72]
39	Spezial Schwartz 4	physical adsorption	avidin	[73]
40	Candle soot	covalent cross-linking with glutaraldehyde	Protein A	[73]

Table 1. Characteristics of CBNP-based detection reagents.

No	CBNP’s Sources	Bioconjugation Method	Recognition Molecule	Ref.
1	Spezial Schwartz 4	physical adsorption	anti-albumin antibody	[10]
2	Spezial Schwartz 4	physical adsorption	anti-alpha-amylase antibody	[43]
3	Spezial Schwartz 4	physical adsorption	monoclonal antibody	[44]
4	N/S	physical adsorption	Mouse anti-rat IgG F(ab′)2 fragment specific antibody	[45]
5	Spezial Schwartz 4	physical adsorption	Anti-IgE immunoglobulin G	[46]
6	N/S	physical adsorption	monoclonal antibodies vs Verotoxin-Producing Escherichia coli	[47]
7	Carbon nanoparticles, in the form of nanostrings, were purchased from Maiia Diagnostics (Uppsala, Sweden)	physical adsorption	anti-influenza A nucleoprotein Monoclonal antibody	[48]
8	carbon black anti-EPO suspension, MAIIA Diagnostics	physical adsorption	anti-EPO antibody	[49]
9	N/S	N/S	anti-SFTSV antibody	[50]
10	Carbon black N115	physical adsorption	Mouse anti-human IgG monoclonal antibody	[51]
11	Carbon black 100	physical adsorption	Dengue Virus NS1 glycoprotein mouse monoclonal antibody	[12]
12	Spezial Schwartz 4	physical adsorption	avidin	[11]
13	Spezial Schwartz 4	physical adsorption	antibody	[52]
14	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[4]
15	N/S	physical adsorption	neutravidin	[53]
16	Spezial Schwartz 4	physical adsorption	neutravidin	[54]
17	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[55]
18	Candle soot	physical adsorption followed by treatment with glutaraldehyde	monoclonal antibody and receptor	[14]
19	Candle soot	physical adsorption followed by treatment with glutaraldehyde	monoclonal antibody	[15]
20	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[56]
21	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[57]
22	Spezial Schwartz 4	physical adsorption	neutravidin	[58]
23	Special blacks and other grades from Degussa AG	physical adsorption	monoclonal antibody	[59]
24	Spezial Schwartz 4	physical adsorption	scCro DNA binding protein	[60]
25	Maiia Diagnostics	physical adsorption	antibiotin antibody or Oligonucleotide	[61]
26	Spezial Schwartz 4	physical adsorption	Polyclonal antibody	[62]
27	Special Black 100	covalent cross-linking by a Silane	monoclonal antibody	[35]
28	Spezial Schwartz 4	physical adsorption	neutravidin	[63]
29	Amorphous CNPs	physical adsorption	Polyclonal antibody	[64]
30	Spezial Schwartz 4	physical adsorption	p48 protein	[65]
31	Spezial Schwartz 4	physical adsorption	neutravidin	[66]
32	Spezial Schwartz 4	physical adsorption	Goat anti-Mouse IgG FcY	[67]
33	Candle soot	covalent cross-linking with glutaraldehyde	monoclonal antibody	[16]
34	Degussa, Düsseldorf, Germany	physical adsorption	polyclonal antibodies	[68]
35	N/S	physical adsorption	monoclonal antibody	[69]
36	Special Black 4	physical adsorption	monoclonal antibody	[70]
37	Candle soot	covalent cross-linking with glutaraldehyde and biotin-streptavidin interaction	DNA-aptemer And biotin	[71]
38	Special Black 100	physical adsorption	monoclonal antibody	[72]
39	Spezial Schwartz 4	physical adsorption	avidin	[73]
40	Candle soot	covalent cross-linking with glutaraldehyde	Protein A	[73]

This type of CB undergoes a specialized post-processing treatment to introduce multiple aldehyde groups. As a result, it does not require a stabilizer in suspension to maintain colloidal stability of the nanoparticles [74]. Consequently, both the preparation of aqueous suspensions and the functionalization of these nanoparticles are straightforward procedures.

The SB4 carbon black nanoparticles possess a very limited number of functional groups, which precludes covalent bonding with proteins or DNA. Nonetheless, physical adsorption offers the advantage of generally preserving specificity. The specific orientation necessary for the display of binding or active domains of antibodies or enzymes in solutions can be achieved by using intermediary biomolecules such as protein A, protein G, or a secondary antibody.

Thus, for SB4 carbon black nanoparticles the simplest, most efficient and fastest method for surface biomodification and simultaneous stabilization of CBNPs with functional proteins is direct physical sorption. As stated in the article [10] the choice of buffer and/or pH for the preparation of colloidal particle-protein conjugates by this method largely depends on the pI of the adsorbed protein. As a rule, the pH of the buffer solution should be 0.5–1.0 pH units higher than the pI of the protein. Usually, a borate buffer with an ionic strength of 5 mM b pH 8.8 is used, since mainly conjugates of SBNP with monoclonal antibodies are described. The ionic strength of the buffer should be maintained at or below 5 mM. To confirm that the suspension has been stabilized, a flocculation test is suggested. The process of obtaining conjugates of carbon nanoparticles with recognition molecules by physical sorption, despite its time efficiency and simplicity, is difficult to control and the reproducibility of the analytical properties of the diagnostic reagents obtained in this way is questionable. None of the articles contain a study of the reproducibility of this synthesis method (

Table 2. Comparison of the CBNPs functionalization methods.

Functionalization Method	Advantages	Disadvantages
Physical adsorption	Fast Efficiency has been demonstrated in various assay formats and across different targets.	The stability of bioconjugates obtained by physical sorption has been poorly studied. The reproducibility of the synthesis method has been poorly studied. Weak binding of recognition molecules to the surface of nanoparticles [75].
Covalent attachment by glutaraldehyde	The functional activity of the conjugates has been demonstrated to be maintained for 10 years or more	Time-consuming The use of this method for creating CBNP-based immunoassays is underrepresented in the scientific literature. The stability of the bioconjugates obtained by this method has not been adequately studied. The reproducibility of the synthesis method has been poorly studied.
Covalent attachment by a Silane	The diversity of silanes allows the choice of the type of chemical bond for surface functionalization of CBNPs according to analytical purposes.	Time-consuming The use of this method for creating CBNP-based immunoassays is underrepresented in the scientific literature. The stability of bioconjugates obtained by this method has not been adequately studied. The reproducibility of the synthesis method has been poorly studied. The advantages of using this method over physical sorption in immunoassay development have not been demonstrated. Poorly controlled adhesion process [76].

).To design analytical platforms using carbon black other than SB4, stabilizers are required [41,42].

Immunoassays are described in which a conjugate based on carbon black CBNP is prepared by covalent attachment using glutaraldehyde (GA). In the articles [16,71] CBNPs from soot is first treated with an inert protein and the bifunctional crosslinking reagent GA to form carboxyl groups. Protein molecules can then be attached to the carboxyl groups via amino groups. This method seems to be more complex and time-consuming due to the stage of GA processing and the need to carry out 2 stages of column chromatography. However, as was shown in the article [16], this method allows obtaining diagnostic reagents that do not lose their colloidal stability and functional activity for 10 years.

Another covalent method for attaching recognition molecules to CBNPs is a method using various silanes [35]. The authors of this study proposed to treat the surface of nanoparticles with 1-pyrenebutanoic acid succinimidyl ester, incubate at 60 °C with silanes (3-mercaptopropyl-trimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane) and stabilize with Atlox acrylic polymer and xanthan dispersing agent. However, the described modification with silanes or the introduction of dispersing agents did not enhance antibody binding or colloidal stability of the diagnostic reagent in the assay.

As shown in Table 1 and Table 2, physical sorption is the most commonly used method for producing CBNP-based diagnostic reagents. This method’s popularity is due to its extensive coverage in the literature and proven effectiveness in detecting various targets. Although, covalent attachment of recognition molecules to the CBNPs surface using glutaraldehyde or silanes modification has been poorly studied and is represented by only a few examples (see Table 1).

The article [45] reported on the conjugation of CBNPs with DNA aptamers by incorporating cadmium chloride and thioacetamide into their composition. This allowed us to introduce functional groups and modify the surface of thiolated aptamer particles. The reaction is carried out in 50 mM Tris–HCl buffer with pH 7.4.

We also note the possibility of modifying the surface of CB nanoparticles with antibodies and dyes using the reaction of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinamide. The authors of the work [77] showed the successful use of such bioconjugates of CBNP with antibodies labeled with horseradish peroxidase to enhance the signal in an immunochemosensor for determining cancer markers. However, the use of these two methods of CBNP functionalization in express assays for visual reading of results has not been shown, and their reproducibility and preservation of properties during long-term storage have not been studied.

3.2. Characterization Method of Bioconjugates Based on CBNPs

Synthesis methods of CB allow obtaining nanoparticles and its bioconjugates with various characteristics in a wide range: size, shape, physical and structural characteristics of the surface; specific surface area, zeta potential and chemical composition of the surface [32,33,78]. All these parameters can influence the application of CBNPs in the design of immunoassays [35,51,74,78,79,80,81].

This review is devoted to visual assessment tests, these are usually lateral-flow immunoassays (LFIA), vertical-flow immunoassays (VFIA), dot-immunoassay and microfluidic tests. Therefore, the most important parameter of CBNPs that must be assessed is the size of the nanoparticles used [51]. The sizes of CBNPs vary depending on the brand, the method of production, the matrix in which the measurements were carried out, the measurement method [79].

The article [79] shows the measurement of CBNPs of three different grades by DLS in a biological media (medium for cell cultures) and in a polymeric dispersant based on styrene and acrylic acid. It was demonstrated that the mean particle diameter increased on average by 2 times when measured in a polymeric dispersant. Summarizing the latest data: the sizes of single CBNPs can range from 10 to 100 (300) nm, while their agglomerates in various environments can reach up to 1000 nm in diameter [82].

The sizes of CBNPs are most often studied using two standard methods: microscopy (SEM, TEM, TEM/AIA (TEM images analyzed with an Automated Image Analysis)) [83,84], AFM [12] and dynamic light scattering (DLS) [85]. Also, in the article [40] it was proposed to distribute nanoparticles and their conjugates by size using flow cytometry.

Another important characteristic when working with CB nanoparticle conjugates is the surface charge (zeta potential). It serves as a parameter of the bioconjugate colloidal stability [79,86].

In 2021, a method for studying aqueous suspensions of CBNPs was proposed, consisting of asymmetrical flow field flow fractionation coupled to UV–vis and DLS detectors in series (AF4-UV–vis-DLS). The proposed method allows one to simultaneously evaluate the size distribution and composition of CBNPs suspensions. Moreover, AF4 allows one to determine the suspension behavior through the separation of different size distributions present in the bulk dispersion and allows quantification studies [79,86].

To evaluate the efficiency of various methods of modification and functionalization of the surface of nanoparticles with recognition molecules, a set of chemical analysis methods is used. The chemical composition of the surface of CBNPs after functionalization is studied using Raman spectroscopy, UV-Vis spectroscopy, electrochemical impedance spectroscopy, thermogravimetric analysis, differential scanning calorimetry, and Fourier transform infrared spectroscopy (FTIR) [87,88]. It should be noted that traditionally, carbon black analysis by IR spectroscopy is difficult due to the high optical density of the material, which remains opaque even with very thin sections. However, modern commercial solutions include the use of IR spectroscopy with attenuated total reflectance spectroscopy (ATR-FTIR), which allows obtaining high-quality spectra of the surface layers of carbon black nanoparticles [89].

X-ray photoelectron spectroscopy (XPS) [77] and energy dispersive X-ray analysis (EDX) [40] were successfully used to study the chemical composition of carbon black nanoparticles and its conjugates.

The molecular weight of bioconjugates of CBNPs with recognition molecules can be determined using gel permeation chromatography, which is described in detail in the study [88].

In the work [90], it was proposed to investigate the structural changes in proteins after conjugation with CBNPs using fluorescence spectroscopy, 3D-fluorescence spectroscopy and the construction of circular dichroism spectra. In addition, Chen et al. studied changes in the functional properties of proteins after binding to nanoparticles using the Protein ANalysis Through Evolutionary Relationships (PANTHER) classification system http://www.pantherdb.org/ (accessed on 30 September 2025) and the functional gene analysis tools of the Database for Annotation, Visualization and Integrated Discovery (DAVID) [40].

4. Use of Diagnostic Reagents Based on CBNPs in Non-Instrumental Immunoassays for Visual Assessment

4.1. Non-Instrumental Immunoassays Based on CBNPs for Visual Assessment

The diagrams of the three most common analysis formats are shown in Figure 1.

Table 3. CBNP-based non-instrumental immunoassay.

No	Assay Format	Scope	Target	Reproducibility of the Diagnostic Reagent Synthesis Method	Stability During Long-Term Storage of the Diagnostic Reagent Synthesis Method	Ref.
1	Dot-immuno assay	Food quality control	Soybean Kunitz-type trypsin inhibitor, human serum albumin mouse immunoglobulin isotyping	N/S in manuscript	N/S in manuscript	[10]
2	LFIA	Food quality control	Fungal alpha-amylase	N/S in manuscript	N/S in manuscript	[43]
3	LFIA	Medicine	Schistosoma circulating cathodic antigen	N/S in manuscript	Remain stable and do not lose functional stability over the course of a year. Numerical values unspecified.	[44]
4	LFIA	Medicine	Sulfamethazine	N/S in manuscript	Retains properties for 6 months in a test strip in a vacuum-sealed bag at room temperature	[45]
5	LFIA	Medicine	IgE	N/S in manuscript	N/S in manuscript	[46]
6	LFIA	Food quality control	Verotoxin-Producing Escherichia coli	N/S in manuscript	N/S in manuscript	[47]
7	LFIA	Medicine		N/S in manuscript	N/S in manuscript	[48]
8	LFIA	Medicine	Erythropoetin	N/S in manuscript	N/S in manuscript	[49]
9	LFIA	Medicine	Thrombocytopenia syndrome virus	N/S in manuscript	N/S in manuscript	[50]
10	VFIA	Medicine	IgG against Spike-protein	Reproducibility confirmed on three batches	Loss of functional activity was noted after one month of storage as a suspension. Numerical values unspecified.	[51]
11	Dot-immunoassay	Medicine	Dengue virus NS1 glycoprotein	N/S in manuscript	N/S in manuscript	[12]
12	LFIA	Geno- typing	E. coli	N/S in manuscript	The signal level in the control line is shown to be maintained for 35 days as part of the test strip.	[11]
13	LFIA VFIA	Food quality control	Hazelnut protein and peanut protein	N/S in manuscript	N/S in manuscript	[52]
14	LFIA	Food quality control	Salbutamol	N/S in manuscript	It was noted that the diagnostic reagent retains functional activity in the composition for 4.5 months. Numerical values unspecified.	[4]
15	Microarray immunoassay with colorimetric detection	Veterinary/ Agriculture	DNA for six different mastitis pathogens	N/S in manuscript	N/S in manuscript	[53]
16	LFIA	Veterinary/ Agriculture	Shiga toxin-producing Escherichia coli virulence factors	N/S in manuscript	N/S in manuscript	[54]
17	LFIA	Food quality control	Forchlorfenuron	N/S in manuscript	N/S in manuscript	[55]
18	Multiplex LFIA	Food quality control	22 β-lactams	N/S in manuscript	N/S in manuscript	[14]
19	Multiplex LFIA	Food quality control	Deoxynivalenol, T-2 toxin, zearalenone	N/S in manuscript	The diagnostic reagent in the test strip was stable and retained functional activity at room temperature for at least 6 months.	[15]
20	LFIA	Environmental safety	Methiocarb	N/S in manuscript	The stability test indicated the strips could be used at least 2 months without change in performance	[56]
21	LFIA	Food quality control	Thiabendazole	N/S in manuscript	The stability test indicated the strips could be used at least 2 months without change in performance	[57]
22	LFIA	Genotyping/Medicine	Genus Cronobacter	N/S in manuscript	The stability test indicated the strips could be used at least 6 months without change in performance	[58]
23	LFIA	Medicine	IgE	N/S in manuscript	N/S in manuscript	[59]
24	LFIA	Genotyping	DNA (PCR-product)	N/S in manuscript	N/S in manuscript	[60]
25	LFIA	Genotyping	DNA	N/S in manuscript	N/S in manuscript	[61]
26	LFIA	Food quality control	Carbaryl	N/S in manuscript	The stability test indicated the strips could be used at least 2 months without change in performance	[62]
27	Dot-immunoassay in biochip-format	Medicine	Histamine	N/S in manuscript	N/S in manuscript	[35]
28	Antibody Microarray assay	Food quality control	Food pathogens	N/S in manuscript	N/S in manuscript	[63]
29	LFIA	Food quality control	Adulteration of cow’s milk with buffalo’s milk	N/S in manuscript	It was noted that the diagnostic reagent retains functional activity for at least a year. Numerical values unspecified.	[64]
30	LFIA	Veterinary/ Agriculture	Mycoplasma bovi	N/S in manuscript	N/S in manuscript	[65]
31	LFIA	Genotyping	DNA (PCR-product)	N/S in manuscript	N/S in manuscript	[66]
32	LFIA	Medicine	Prostate specific antigen	N/S in manuscript	N/S in manuscript	[91]
33	LFIA	Environmental safety	Chlorpyrifos	N/S in manuscript	N/S in manuscript	[67]
34	LFIA VFIA Dot-imunoassay	Medicine	Human chorionic gonadotropin, Immunoglobullin, HIV protein	N/S in manuscript	The stability test indicated the diagnostic reagent could be used at least 10 years without change in performance	[16]
35	LFIA	Medicine	Human chorionic gonadotropin	N/S in manuscript	N/S in manuscript	[69]
36	LFIA	Medicine	Extracellular vesicles	N/S in manuscript	N/S in manuscript	[70]
37	Dot-imunoassay	Medicine	IgE	Reproducibility confirmed on three batches	The stability test indicated the diagnostic reagent could be used at least 30 days without change in performance	[71]
38	Dot-imunoassay in a microarray format	Medicine	Cancer biomarkers	N/S in manuscript	N/S in manuscript	[72]
39	LFIA	Medicine	DNA (amplicon)	N/S in manuscript	N/S in manuscript	[73]
40	Dot-imunoassay	Medicine	Group A streptococcal antibodies	N/S in manuscript	N/S in manuscript	[92]

lists the existing diagnostics tests based on CBNPs. The selection includes studies describing the development of test systems using CBNPs that provide visual, non-instrumental assessment of results. In most studies, CBNP-based diagnostic reagents were used to create LFIA (Table 3, No 2–9, No 12–26, No 29–36, 39]. In addition, other non-instrumental test formats are considered, such as VFIA (Table 3, No 10, 13, 34), dot-immunoassays [1,11,34,37], and dot-immunoassays in the microarray format (Table 3, No 28, 38). Also, the format of analyses based on CBNPs can be multiplex (Table 3, No. 18, 19).

The first scientific article demonstrating the prospects for using CBNPs in the development of rapid immunochemical tests with visual assessment was published in 1993 [10]. This work describes the creation of a dot-immunoassay for the simultaneous determination of several biomarkers using CBNPs.

Currently, test systems with visual assessment of results have been described, used in medicine, food industry, environmental monitoring, veterinary science and genotyping (Figure 2). The developed analytical methods are capable of identifying a wide range of targets-from DNA and proteins to extracellular vesicles (Table 3).

As shown in Table 3, most studies generally do not give sufficient attention to assessing the reproducibility of synthesis methods and the stability of diagnostic reagents based on CBNPs. Most published studies do not systematically analyze the stability of carbon nanoparticles conjugates with recognition molecules and the preservation of their functional properties during long-term storage. 32% of the reviewed studies mention storage conditions and periods with an assessment of changes in the functional characteristics of CBNPs modified with recognition elements (Figure 2). However, only 22% of publications provide such data with numerical indicators or visual materials (photographs, scans, etc.). Only one study assessed the preservation of the functional properties of diagnostic reagents for a period exceeding one year, presenting the results in the form of test photographs [12].

In addition, only two studies assessed the reproducibility of the method of functionalization of nanoparticles with recognition molecules [38,49]. However, the analysis of only three batches of conjugates is insufficient for full statistical processing and the formation of confident conclusions about the reproducibility of the described methods for obtaining diagnostic reagents.

The described gaps will be a significant drawback when implementing CBNPs in real practice.

4.2. CBNPs in Assay Design Compared to Other Labels

Traditionally, in non-instrumental assays with visual assessment, colloidal gold nanoparticles are used as a color label [8,93]. For this reason, in most studies on the design of test systems, the efficiency of diagnostic reagents of the CBNPs-recognizing molecule is compared with the functional activity of those based on colloidal gold (

Table 4. Comparison of CBNPs with other colorimetric labels.

Influence	Type of Nanoparticles for Comparison	Numerical Indicators of Effect	Ref.
	(1) Silver coated gold nanoparticles, gold nanoparticles and polystyrene beads	Carbon black had a low detection limit of 0.01 μg/mL in comparison to 0.1 μg/mL, 1 μg/mL and 1 mg/mL for silver-coated gold nanoparticles, gold nanoparticles and polystyrene beads	[12]
	(2) gold nanoparticles	LOD for visual detection of as low as 2.2 × 10−2 pg μL−1 for CBNPs and 8.4 × 10−2 pg μL−1 for AuNPs	[11]
Analytical performance improvement	(3) gold nanoparticles	CBNPs provided a higher sensitivity with 2-fold lower cut-off values than that of colloidal gold labels	[14]
	(4) gold nanoparticles and quantum dots	The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs.	[15]
	(5) enzymes: b-galactosidase and peroxidase	The detection limit was for b-galactosidase 0.3 amol and for horseradish peroxidase 5 amol enzyme/microplate well. While the detection limit for carbon black was decreased to 0.02 amol /mm2 membrane	[59]
Analytical performance decreased	(1) Colloidal gold and nanogold-polyaniline-nanogold microspheres	For the nanogold-polyaniline-nanogold microspheres and colloidal carbon, the limit of detection was 10 µg kg−1 in meat samples vs. 5.0 µg kg−1 for the colloidal gold	[4]
	(2) Colloidal gold and magnetic nanoparticles	LOD for CBNPs (9.25 × 106 EVs/μL) significantly lower in comparison with MNPs (1.05 × 106 EVs/μL). At the same time, CBNPs are inferior in sensitivity for gold NPs (LOD = 4.55 × 106 EVs/μL)	[70]
No effect	(1) Horseradish peroxidase, CBNPs with Horseradish peroxidase modification	The limits of detection were between 6.9 and 10.4 ng of PCR product for all three labels	[60]
	(2) Colloidal gold and fluorescent dye	The LOD for CBNPs for were comparable with LOD for colloidal gold and fluorescent dye	[35]

).

The studies [11,12,14,15] demonstrated an increase in sensitivity when using CBNPs for the synthesis of a diagnostic reagent in comparison with gold ones. For example, in the article [12], using CBNPs, it was possible to reduce the lower limit of detection (LOD) by 100 times for the biotin-streptavidin system (from 1 μg/mL for gold nanoparticles, to 0.01 μg/mL for carbon black nanoparticles). Moreover, the authors found that the colorimetric detection limit of 57 ng/mL of Dengue virus nonstructural glycoprotein for carbon black was ten times lower than the 575 ng/mL observed for standard gold nanoparticles, which makes it sensitive enough to diagnose a patient on the first days of infection [12].

On the other hand, in works [4,70] the functional activity of conjugates based on CBNPs was lower in comparison with colloidal gold nanoparticles.

Also, one of the traditional labels for creating diagnostic reagents are enzymatic labels (mainly horseradish peroxidase). There are also contradictory data on the comparison of the efficiency of using CB nanoparticles with this type of labels. In the article [59], the LOD of the analysis when using CBNPs decreases compared to b-galactosidase and peroxidase. However, the authors of the work [60] did not confirm a significant increase in sensitivity when working with CB nanoparticles in comparison with horseradish peroxidase.

The observed differences in the results of studies conducted by various research teams are most likely due to the fact that the methods of functionalization with recognition molecules differ depending on the type of label. In the reviewed articles, CBNP-based conjugates were obtained by physical sorption, while conjugates with horseradish peroxidase were obtained using the Nakane method [94]. Moreover, CBNPs-based conjugates for comparative studies were prepared directly in the laboratory, whereas gold nanoparticle-based diagnostic reagents were obtained from various commercial suppliers, with the supplier companies differing across manuscripts. Accordingly, the functional activity of the compared diagnostic reagents used in different studies may also vary. The analysis format could have also influenced the comparison results. For example, in articles where carbon nanoparticles increased the sensitivity of the method compared to gold nanoparticles, the evaluation was conducted using immunochromatography. While the article describing the decrease in sensitivity with CBNPs was evaluated using a dot immunoassay, the results were inconsistent. In general, as noted by Farka et al. in their 2024 review, comparing individual assay parameters has limited value for evaluating the overall performance of an assay, since its success depends on the overall concept of the technology platform [1].

The inconsistent data obtained by different research groups is also due to the fact that bioconjugates based on traditional labels (latex and gold nanoparticles, enzyme labels) are currently much more widely used. This means that the methods for producing diagnostic reagents based on them are using these labels are well-optimized and thoroughly studied. In contrast, bioconjugates based on CBNPs have been less well-studied—very little data is available on the reproducibility of the functionalization method and the optimal conditions for their implementation. This may ultimately lead to the production of CBNP-based diagnostic reagents with varying performance in immunoassays. Although some studies report that CBNPs do not significantly increase sensitivity compared to traditional labels, improvements in other analytical parameters are demonstrated. The study [35] demonstrated that a colorimetric assay employing oxidized CBNPs achieved a wider dynamic range (44–293 μg/mL) than a fluorescent assay (35–236 μg/mL). Furthermore, the research presented in [60] reported that diagnostic reagents based on CBNPs not only extended the dynamic range but also eliminated nonspecific background signals, in comparison to enzyme-based labels.

Table 4 shows that most studies report better analytical performance for assays based on CBNPs compared to those using other labels. This improvement is mainly attributed to CBNPs large surface area, ease of functionalization, and strong visual contrast. Additionally, many sources highlight the low cost of producing carbon black. For instance, 1 g of N220-grade CBNPs costs approximately USD 0.0001, while 1 g of gold nanoparticles (20–60 nm) costs USD 430–435, and 1 g of latex nanoparticles costs USD 1120. This cost difference is particularly important when resources are limited [95,96,97], which is especially relevant when resources are limited.

5. Conclusions: Future Perspectives and Recommendation

CBNPs continue to represent a promising alternative to traditional nanoparticles. Numerous studies comparing the analytical performance of newly developed methods indicate that using CBNPs as color labels improves key parameters such as the limit of detection and the dynamic linear range.

However, it is important to note that commercially available diagnostic products incorporating CBNPs conjugated with recognition molecules are currently nonexistent. The sole exception is the nanorings developed by Maiia Diagnostics [98]. The precise reasons for the low availability of CBNP-based diagnostic reagents on the laboratory diagnostics market are difficult to determine. From a scientific perspective, these include the lack of comprehensive studies on the reproducibility of CBNP-based diagnostic reagent production methods, as well as the paucity of studies assessing their stability during long-term storage (more than one year) and the associated changes in their analytical performance. On the other hand, the complexity of this issue is that it is not limited solely to the scientific aspect. Commercial success requires not only competitive technology, but also financial resources, business experience, and timely patent filing. This applies not only to CB nanoparticles but to nanomaterials in general [1].

Another possible reason for the difficulties in bringing CBNPs bioconjugates to market and into practical use is the lack of a unified regulatory framework governing the production, preparation, use, and disposal of nanomaterials [99]. Most existing regulations are designed for bulk chemicals and are ill-suited for their nanoanalogues. Despite ongoing efforts by regulatory agencies to standardize information on nanoproducts, the path to full harmonization remains very long.

Future research should emphasize the following recommendations. Current studies do not adequately assess the reproducibility and stability of CBNPs reagents. Therefore, when developing new procedures for synthesizing CBNPs functionalized with recognition molecules and designing test systems based on these bioconjugates, it is essential to provide detailed data on the reproducibility of synthesis methods. Additionally, the stability of these bioconjugates during storage should be thoroughly evaluated, both as suspensions and within test systems—for example, in dried form on the conjugate pad of immunochromatographic test strips.

Research focused on developing test systems using various brands of CBNPs other than Special Black 100 and Spezial Schwartz 4, as well as comparing the characteristics of the resulting diagnostic reagents and analytical methods, may also be promising.

It is important to note that most functionalized colloidal carbon nanoparticles modified with recognition molecules are obtained by direct physical sorption (Table 1). In this regard, the study of alternative methods for the functionalization of CBNPs continues to be an urgent task.

A weak point in attempts to introduce analytical methods based on CBNPs into real practice and their production on a large scale may be their potential negative impact on health [100,101,102,103]. Therefore, new research in this direction is also necessary.