Trends and frontiers in signal amplification for aptamer-based tumor detection: A bibliometric analysis

Abstract

BACKGROUND

Malignant tumors are one of the leading causes of death worldwide, imposing a substantial economic and social burden. Early detection is the key to improving cure rates and reducing mortality rates, which requires the development of sensitive early detection technologies. Signal amplification techniques play a crucial role in aptamer-based early detection of tumors and are increasingly garnering attention from researchers.

AIM

To investigate the current research status, developmental trajectories, and hotspots in signal amplification for aptamer-based tumor detection through bibliometric analysis.

METHODS

English publications pertaining to signal amplification in aptamer-based tumor detection were retrieved from the Web of Science Core Collection database. VOSviewer and CiteSpace software were employed to analyze various information within this field, including countries, institutions, authors, co-cited authors, journals, co-cited journals, cited references, and keywords.

RESULTS

A total of 757 publications were included in this study. China accounted for 85.47% of all publications, with Nanjing University (China) emerging as the institution with the highest publication output. The most influential authors and journals were Hasanzadeh M. from Iran and "Biosensors and Bioelectronics", respectively. Exosomes and carcinoembryonic antigen (CEA) stood out as the most researched tumor-related molecules. Currently, the predominant signal amplification technique, nanomaterial, and signal transduction method were identified as hybridization chain reactions, gold nanoparticles, and electrochemical methods, respectively. Over the past 3 years, exosomes, CEA, electrochemical biosensors, and nanosheets have emerged as research hotspots, exhibiting a robust burst of intensity.

CONCLUSION

This study is the first bibliometric analysis of literature on signal amplification in aptamer-based tumor detection and elucidates the current status, hotspots, and prospective research directions within this realm. Additionally, it provides an important reference for researchers.

Key Words: Aptamer; Signal amplification; Tumor; Bibliometrics; Electrochemistry; Nanosheet

Core Tip: This study is the first bibliometric analysis of the literature pertaining to signal amplification in aptamer-based tumor detection. It unveils that China accounts for 85.47% of all publications, with Hasanzadeh M of Iran and the journal "Biosensors and Bioelectronics". Furthermore, it highlights exosomes and carcinoembryonic antigen as the most extensively studied tumor-related molecules.

INTRODUCTION

Malignant tumors are one of the leading causes of death worldwide, imposing a substantial economic and social burden[1]. Achieving early detection is key to improving cure rates and reducing mortality rates, which requires the development of sensitive early detection technologies[2]. Nucleic acid aptamers, acquired via systematic evolution of ligands by exponential enrichment technology, can bind to their targets with remarkable specificity and affinity. Functioning akin to antibodies, they offer advantages including lower cost, smaller size, facile chemical modification, and reversible target binding[3]. The utilization of aptamer-based sensors has witnessed rapid advancements in the field of tumor detection[4,5].

However, the performance of aptamer-based sensors in detecting low-abundance targets is poor. Unfortunately, tumor biomarkers often exhibit low abundance, significantly limiting the utility of aptamer-based sensors in early tumor detection. Therefore, many strategies have been devised to enhance signal amplification, such as hybridization chain reaction (HCR)[6,7], enzyme-based amplification[8], and nanomaterial amplification[9]. These approaches hold great promise for enabling early and highly sensitive tumor detection, thus poised to emerge as pivotal tools for tumor detection.

Bibliometric analysis is a novel statistical approach aimed at delineating the current state of research, trends, and frontiers within a specified research field by quantitatively analyzing the characteristics and structure of the literature in that field[10,11]. VOSviewer and CiteSpace software are widely used for performance analysis, elucidating the contributions of research components, as well as visualization mapping, delineating the interrelations among research components[12]. Consequently, these approaches provide insights and avenues for researchers. Currently, bibliometric analysis has made significant contributions in the fields of tumor etiology[13], tumor metastasis[10], and tumor treatment[14].

Nonetheless, no bibliometric research has been conducted on signal amplification in aptamer-based tumor detection. In this study, a bibliometric analysis was performed to delineate the history and current status of signal amplification strategies in aptamer-based tumor research. The analysis aimed to uncover research trends, identify hotspots, delineate future directions, and provide references for researchers interested in this field.

MATERIALS AND METHODS

Literature acquisition

The Web of Science Core Collection (WoSCC) is renowned as one of the most comprehensive and authoritative academic databases, often employed for bibliometric analysis[15]. The research data for this study were acquired via a term search of this database, utilizing the following terms: TS = ("aptamer" OR "aptamers" OR "aptasensor" OR "aptasensors" OR "aptasensing" OR "aptazyme" OR "aptazymes") AND TS = ("signal amplification" OR "signal amplifications" OR "signal-amplified" OR "signal amplifier" OR "signal amplifiers" OR "signal amplifying" OR "signal amplifyings" OR "signal amplifiable" OR “amplified” OR “cycling” OR “chain reaction” OR “enzyme assisted”) AND TS = ("carcinoma" OR "carcinomas" OR "cancer" OR "cancers" OR "tumor" OR "tumors" OR "neoplasm" OR "neoplasms").

During the search, the publication period was set to January 2010 to July 2023, and the publication type was set to articles and reviews. All literature was exported and archived in plain text format, in the form of full records and referenced citations. The search process and subsequent screening were performed independently by two authors. Disagreements were resolved through discussion or, if necessary, the intervention of a third author. A total of 757 eligible articles were included in this study. Figure 1 illustrates the search and screening process.

Figure 1 Flowchart of literature search and screening. WoSCC: Web of Science Core Collection.

Literature analysis

The VOSviewer (V.1.6.19) and CiteSpace (V.6.2.4) software were employed for the analysis and visualization of various research components, including countries, institutions, authors and co-cited authors, journals and co-cited journals, co-cited references, and keywords. In addition, CiteSpace was employed to analyze dual-map overlays, co-cited reference clustering, burst citations, and keyword burst citations.

In data analysis visualization maps, each node represents an analyzed research component, with its size proportionate to the frequency of occurrence. The interconnections among these nodes illustrate the co-occurrence relationships between research components, with the thickness of connections contingent upon the frequency of such co-occurrences[12]. Total publications, total citations, average citations, and the H-index were employed to assess the influence of research elements. The H-index denotes the number of publications garnering at least h citations for h publications published by a specific research element. These metrics offer insights into both the overall and individual impact of research components[12,16].

RESULTS

Annual publications and citations

Annual publication and citation analyses offer insights into the developmental trajectory of a given research field[17]. Figure 2 illustrates the yearly trends in both publications and citations within the literature concerning signal amplification in aptamer-based tumor detection, spanning from January 2010 to July 2023. Signal amplification for aptamer-based tumor detection was first reported in 2010. Before 2013, this field was in its infancy, characterized by slow growth. From 2014 to 2018, a phase of rapid growth ensued, marked by an exponential rise in the number of annual publications, indicating that the significance of signal amplification for aptamer-based tumor detection was increasingly being recognized by researchers. Since 2018, the field seems to have stabilized, albeit with fluctuations in annual publication figures. Compared to the number of publications, the number of annual citations has steadily increased, suggesting that the field continues to be of interest among researchers.

Figure 2 Annual counts of publications and citations from 2010 to 2023.

Analysis of countries, institutions, and their collaborations

A total of 757 publications emanated from 594 institutions spanning 39 countries. The ranking of the top 10 countries and institutions based on publication count is shown in Table 1. China led with the highest number of publications, accounting for 85.47% of the total publications, followed by Iran (7.01%) and the United States (5.55%). Citations and H-index exhibited a similar trend. It is worth highlighting that although Singapore (rank 11) published only five articles, its influence was significant, with average citations of 219.2. Among institutions, the top 10 entities with the highest publication counts all hail from China, collectively contributing 40.4% (n = 306) of the total publications. Furthermore, these institutions boasted an H-index of ≥ 10, underscoring their substantial influence in this field.

Table 1 Top 10 countries and institutions ranked by publication counts.

Rank	Country/institution	Publications	Citations	Average citations	H-index
	Country
1	China	647	20896	32.3	77
2	Iran	53	2514	47.4	30
3	United States	42	1753	41.7	26
4	Spain	13	636	48.9	11
5	South Korea	11	437	39.7	6
6	Japan	7	142	20.3	5
7	Canada	6	274	45.7	6
8	England	6	256	42.7	4
9	Turkey	6	235	39.2	5
10	India	6	83	13.8	5
	Institution
1	Nanjing University	45	2290	50.9	27
2	Chinese Academy of Sciences	40	1605	40.1	21
3	Southwest University	38	1230	32.4	22
4	Qingdao University Science and Technology	36	1247	34.6	22
5	Hunan University	33	1373	41.6	20
6	Central South University	32	1089	34.0	24
7	Nanjing Medical University	22	589	26.8	12
8	Chongqing Medical University	21	548	26.1	11
9	Shanghai University	20	753	37.7	15
10	Sun Yat-Sen University	19	678	35.7	13

Collaboration serves as a pivotal means to promote efficient and high-quality research endeavors. The examination of collaboration among countries in the field of signal amplification for aptamer-based tumor detection is depicted in Figure 3A. The most pronounced collaborations emerged between China and the United States, as well as between Iran and Spain. With reference to the color bar of the timeline, it is evident that the bulk of collaborative papers (green nodes) were published around 2019, and a few collaborative articles (yellow nodes) were published in the past 2 years. This trend suggests a relatively recent commencement of collaboration among countries in this field. Figure 3B illustrates the collaboration network among institutions with more than five publications (n = 63). Notably, the collaboration among institutions was more extensive than the collaboration among countries. Moreover, close collaborations were observed between the Chinese Academy of Sciences and various institutions across different provinces of China.

Figure 3 Collaboration networks on the signal amplification for aptamer-based tumor detection. A: National collaboration network map; B: Institutional collaboration network map.

Analysis of authors and co-cited authors

When two publications co-occur as references in another publication, they are deemed to exhibit a co-citation relationship[18]. Analyzing the authors and their co-cited authors within a given field can provide a succinct overview of representative scholars in that domain. Co-cited authors are those whose works are referenced concurrently by another publication, and the frequency of such joint citations indicates their significance. This study encompassed 3282 authors who contributed to research concerning signal amplification for aptamer-based tumor detection. Table 2 presents the top 10 authors with the highest publication counts, among whom 9 hail from China, while the remaining individual hails from Iran. Yuan R emerged prominently with the highest publication count, substantially surpassing Xiang Y and Yang MH, who secured the second rank. The most frequently co-cited author was Hasanzadeh M, followed by Wang J and Zhang Y, with citation counts of 221, 121, and 113, respectively. Employing Price's Law and VOSviewer, we found that there existed 80 core authors (each with five or more publications) who collectively published 584 articles (77.1%). This underscores the establishment of a relatively stable group of core authors in this field.

Table 2 Top 10 authors and co-cited authors ranked by publication and citation counts.

Rank	Author	Counts	Average citations	Country	Co-cited author	Citations
1	Yuan R	33	32.3	China	Hasanzadeh M	221
2	Xiang Y	19	23.2	China	Wang J	121
3	Yang MH	17	33.7	China	Zhang Y	113
4	Wang KM	16	33.2	China	Bi S	102
5	Li GX	14	43.4	China	Wang Y	95
6	Hasanzadeh M	13	56.6	Iran	Li J	87
7	Zhang SS	11	51.5	China	He Y	86
8	Yu JH	10	28.3	China	Ellington AD	84
9	Sun DP	10	50.3	China	Li Y	84
10	Ding SJ	10	56.4	China	Zhang J	83

Figure 4A depicts the network map, illustrating collaboration among the core authors. It is evident that the connections among individual authors are significantly weaker than those between countries and institutions, indicating that current research is primarily focused on individuals and representative groups rather than large-scale academic collaboration. Therefore, enhancing communication and collaboration among teams is necessary to facilitate more expansive and profound research endeavors in this field.

Figure 4 Collaboration networks on the signal amplification for aptamer-based tumor detection. A: Core author collaboration network map; B: Network map of co-cited authors with more than 20 citations; C: The dual-map overlay of journals.

Figure 4B depicts the network relationship among co-cited authors with more than 20 citations. The visualization illustrates these authors grouped into clusters, wherein the node representing Hasanzadeh M exhibits connections with most of the other nodes and intersects with the other three clusters. This indicates that this author may have established the research framework pivotal for the advancement of this field.

Analysis of journals and co-cited journals

The 757 articles originated from 108 distinct journals. Co-cited journals are defined as articles stemming from two journals that were cited jointly by another article. Table 3 delineates the top 10 journals by publication count and co-citation frequency (all positioned in Q1 or Q2 of the journal citation reports). "Biosensors and Bioelectronics" and "Analytical Chemistry" emerged as the foremost journals in terms of both publications and citations, while "Sensors and Actuators. B, Chemical" and "Journal of the American Chemical Society" secured the third position. These journals are esteemed within the field of signal amplification for aptamer-based tumor detection. These findings hold potential utility for scholars in the field as they prepare their scholarly contributions.

Table 3 Top 10 journals and co-cited journals ranked by publication and co-citation counts.

Rank	Journal	Article count	IF (2023)	Co-cited journal	Citation	IF (2023)
1	Biosensors and Bioelectronics	105	12.6	Biosensors and Bioelectronics	4879	12.6
2	Analytical Chemistry	93	7.1	Analytical Chemistry	4725	7.1
3	Sensors and Actuators. B, Chemical	66	8.4	Journal of the American Chemical Society	1458	15.0
4	Analytica Chimica Acta	51	6.2	Sensors and Actuators. B, Chemical	1373	8.4
5	Talanta	45	6.1	Chemical Communications	1204	4.9
6	Microchimica Acta	43	5.7	Angewandte Chemie- International Edition	974	16.6
7	Analyst	29	4.2	Analytica Chimica Acta	909	6.2
8	Chemical Communications	26	4.9	ACS Applied Materials and Interfaces	853	9.5
9	ACS Applied Materials and Interfaces	19	9.5	Talanta	827	6.1
10	Trends in analytical chemistry: TRAC	13	13.1	Analyst	802	4.2

IF: Impact factor; ACS: American chemical society; TRAC: Trends analytical chemistry.

Figure 4C vividly illustrates the effective collaboration across various fields of publications and cited literature. The left panel displays the citing journals, whereas the right panel depicts the cited ones. Two important purple citation paths indicated that the journals Chemistry, Materials, Physics and the journals Molecular, Biology, Genetics were often cited by the journals Physics, Materials, Chemistry. Therefore, signal amplification for aptamer-based tumor detection is closely associated with materials science, chemistry, and medicine, and enhancing multidisciplinary management emerges as a pivotal imperative for future endeavors.

Analysis of co-cited references

Co-cited references refer to two publications cited together by another publication[18,19]. Analyzing co-cited references can illuminate core documents in specific fields and evolutionary trends in research focus[20]. Table 4 presents the top 10 co-cited references, comprising predominantly original articles with the exception of the ninth entry. Ranks 1, 2, 4, and 8 were associated with aptamer selection, while ranks 3 and 9 pertained to HCR. The remaining references predominantly explored aptamer sensors based on nanomaterials for tumor research, which demonstrates that aptamer selection is the foundation of aptamer-based sensors for tumor research, the prominence of the HCR technique, and the burgeoning interest in aptamer sensors based on nanomaterials.

Table 4 Top 10 co-cited references ranked by citation counts.

Rank	Co-cited reference	Citations	DOI	IF (2023)
1	Ellington AD, 1990, Nature, v346, p818	77	10.1038/346818a0	64.1
2	Tuerk C, 1990, Science, v249, p505	70	10.1126/science.2200121	56.9
3	Dirks RM, 2004, Proc Natl Acad Sci USA, v101, p15275	50	10.1073/pnas.0407024101	11.1
4	Shangguan D, 2006, Proc Natl Acad Sci USA, v103, p11838	49	10.1073/pnas.0602615103	11.1
5	Wang S, 2017, Acs Nano, v11, p3943	35	10.1021/acsnano.7b00373	17.1
6	He Y, 2012, Nanoscale, v4, p2054	33	10.1039/c2nr12061e	6.7
7	Zheng TT, 2014, J Am Chem Soc, v136, p2288	32	10.1021/ja500169y	15.0
8	Tan WH, 2013, Chem Rev, v113, p2842	32	10.1021/cr300468w	62.1
9	Bi S, 2017, Chem Soc Rev, v46, p4281	32	10.1039/c7cs00055c	46.2
10	Jiang Y, 2017, Angew Chem Int Edit, v56, p11916	32	10.1002/anie.201703807	16.6

DOI: Digital object identifier; IF: Impact factor.

A cluster map of co-cited references was generated using CiteSpace, as shown in Figure 5A, revealing 10 significant clusters characterized by high Q-values (Q = 0.784) and silhouette scores (S = 0.8779), demonstrating good clustering patterns and high reliability. The largest cluster was "#0 exosomes" with most of the literature pertaining to the detection of tumor-derived exosomes[21-23]. Regarding temporal distribution (Figure 5B), clusters "#0 exosomes" and "#3 carcinoembryonic antigen" are positioned towards the far right end of the timeline, predominantly represented by yellow and orange nodes, indicating their current prominence as focal points of research within the field.

Figure 5 Co-cited references analysis of signal amplification for aptamer-based tumor detection. A: Clustering map; B: Clustering temporal evolution; C: Top 10 references with the strongest citation bursts.

The high citation burst strength of references can reflect frontiers in a given field. Figure 5C illustrates the top 10 references exhibiting the highest citation burst strength, ranging from 6.35 to 8.91. Citations with high burst intensity (denoted by red squares) during 2013–2019 (first five) and 2020–2023 (last five) corresponded to tumor cell surface-associated proteins, particularly Mucin 1, and exosome detection, respectively. Among them, "Huang RR, 2019, SMALL"[22], "Huang RR, 2020, NANOSCALE"[24], and "Zhao L, 2019, ANAL CHEM"[25] were closely associated with signal amplification in exosome detection, while "Kalluri R, 2020, SCIENCE"[26] reviewed the application of exosomes in biomedical fields. This indicates a shift in the researchers' focus from detecting tumor cell surface-associated proteins to achieving precise detection of tumor-derived exosomes.

Analysis of keywords

Keywords typically indicate the central theme of a publication. We analyzed the keywords, after merging synonymous terms, to identify the primary research direction in signal amplification for aptamer-based tumor detection.

Using VOSviewer, we identified 20 high-frequency keywords (Table 5) and found that signal-amplified aptamer sensors have been used to detect various tumor-related biomarkers, such as tumor-derived exosomes, soluble tumor markers, cancer cells, and tumor surface-related proteins, with exosomes and carcinoembryonic antigen (CEA) emerging as the most frequently detected entities. Furthermore, we found that three keywords (ranked 2^nd, 9^th, and 19^th) were associated with electrochemical methods. In addition, the keywords associated with signal amplification methods included "gold nanoparticles" (GNPs) (rank 6), "hybridization chain reaction" (rank 8), "nanomaterial" (rank 17), and "DNAzyme" (rank 18), indicating that the most frequently used signal amplification methods in this field were nanomaterials (especially GNPs), HCR, and DNAzyme-based amplification. Using CiteSpace software, we analyzed the corresponding years of the keywords associated with signal amplification techniques, yielding insightful results: DNAzyme-based amplification (2010), rolling circle amplification (2012), HCR (2014), and catalytic hairpin assembly (2015). On the basis of the aforementioned results, we found that, currently, nanomaterials, particularly GNPs, are most frequently employed in signal amplification in aptamer-based tumor detection research, with HCR emerging as the most frequently used technique for nucleic acid signal amplification.

Table 5 Top 20 author keywords ranked by frequency.

Rank	Keyword	Count	Rank	Keyword	Count
1	Aptamer	133	11	Mucin 1	27
2	Electrochemical biosensor	79	12	Biomarker	26
3	Signal amplification	64	13	Cancer cells	24
4	Biosensor	50	14	Cancer	17
5	Aptasensor	48	15	Tumor markers	17
6	Gold nanoparticles	42	16	Circulating tumor cells	16
7	Exosomes	37	17	Nanomaterial	16
8	Hybridization chain reaction	37	18	DNAzyme	15
9	Electrochemistry	35	19	Electrochemiluminescence	15
10	Carcinoembryonic antigen	29	20	Fluorescence	14

Figure 6A displays a network map of keywords that have appeared more than five times, encompassing 68 keywords and forming three clusters. These clusters, arranged in descending order of size, were "electrochemical biosensor", "aptamer", and "biomarker". Figure 6B shows the top 10 keywords with the strongest citation burst strength. GNPs emerged as the earliest burst keyword, maintaining its prominence from 2010 to 2016. Conversely, recent burst keywords over the past 3 years included "nanosheets", "carcinoembryonic antigen", and "electrochemical biosensors". This suggests a potential shift in researchers' focus within the field from GNPs to nanosheets, while concurrently intensifying attention towards CEA and electrochemical analysis.

Figure 6 Keyword analysis of signal amplification for aptamer-based tumor detection. A: The collaboration network of keywords; B: Top 10 keywords with the strongest citation bursts.

DISCUSSION

The importance of early detection of tumors is undeniable; however, the scarcity of tumor markers poses a significant challenge to achieving this goal. Thus, the development of ultrasensitive, specific, and cost-effective approaches for detecting tumor-related molecules has emerged as a paramount concern in tumor research. Nucleic acid aptamers are innovative molecular diagnostic tools characterized by unique properties that facilitate the highly sensitive detection of targets with low abundance through various signal amplification techniques.

General information

In this study, we conducted a bibliometric analysis of the literature pertaining to signal amplification for aptamer-based tumor research, spanning the period from 2010 to July 2023. A total of 757 articles from 108 journals were analyzed, and these articles were published by 594 institutions across 39 countries, involving a collective of 3282 authors and 24689 co-cited references.

The statistical analysis of annual publications and their citations consistently indicated an upward trend in both the number of publications and citations in this field, underscoring a sustained interest in the subject over the past years. China emerged as the frontrunner in terms of publication output (647 publications), followed by Iran (53 publications) and the United States (42 publications). The total citations and H-index of these publications. The top 10 institutions in terms of publication count were from China, with Nanjing University at the forefront. Yuan R holds the highest publication count (33), while Hasanzadeh M has garnered the most citations (221). While collaborations among various countries and institutions were observed, there remains untapped potential for further collaboration among researchers. The two journals with the most publications and citations in this field are "Biosensors and Bioelectronics" and "Analytical Chemistry". Ranking third in terms of the number of publications and citations are "Sensors and Actuators. B, Chemical" and "Journal of the American Chemical Society" respectively.

Research directions and hotspots

Three of the top four most cited references[27-29] pertain to aptamer selection strategy (Table 4), representing pioneering works in the field of aptamers. The capacity of aptamers to bind to their targets significantly impacts the sensitivity, specificity, and stability of assays. Consequently, since the emergence of aptamers in 1990, researchers have sustained a robust interest in refining the selection or modification of aptamers to enhance tumor detection efficacy[27,30,31].

The timeline map of cited references (Figure 5B) illustrates that, in recent years, highly cited references primarily pertain to the detection of exosomes and CEA, indicating a growing emphasis on the potential applications of exosomes and CEA in tumor research. Exosomes, which are small vesicles released by cells, including cancer cells, serve as carriers of proteins, nucleic acids, lipids, and other molecules, thereby facilitating the transmission of rich molecular information between cells. A comprehensive exploration of exosomes can unveil the mechanisms of cellular communication and the molecular processes underlying diseases[26]. Owing to their significance in early diagnosis, monitoring metastasis, assessing prognosis, and personalized therapies for tumors, as well as their stability and ease of access, exosomes represent ideal liquid biomarkers[32,33] and are rapidly gaining ground in tumor research[34]. Unfortunately, exosome concentration remains low during the initial phases of tumors, and conventional analytical methods, such as western blot[35] and enzyme-linked immunosorbent assay[36], are plagued by limitations such as low sensitivity, high sample volume requirements, and protracted analysis duration. Therefore, in recent years, researchers have endeavored to design and develop signal amplification techniques for the aptamer-based ultrasensitive detection of exosomes[22].

In terms of the most pronounced reference citation bursts (Figure 5C), in the field of aptamer-based tumor detection, earlier investigations (2013–2019) primarily focused on tumor cell surface-associated proteins. Conversely, recent studies (2020–2023) shifted their emphasis towards exosomes. Despite the importance of detecting tumor cell surface-associated proteins for understanding the mechanisms of tumorigenesis, progression, diagnosis, and treatment, its application in liquid biopsy is constrained by the scarcity and heterogeneous nature of circulating tumor cells[32]. In contrast, the biomedical significance of tumor-derived exosomes is becoming more prominent, with exosome detection based on the aptamer strategy poised to remain a pivotal trajectory in future tumor research. The precise detection approach of tumor-derived exosomes is expected to improve the accuracy of early diagnosis, monitoring of disease progression, and evaluation of treatment response, thus providing patients with a more tailored treatment regimen.

However, several challenges hinder the realization of this goal. Firstly, researchers must develop more sensitive, stable, and specific exosome assays to reduce background interference and improve detection sensitivity. Secondly, there is a pressing need to advance precise, time-efficient, and cost-effective techniques for exosome isolation and single-exosome analysis to further investigate the heterogeneity of exosomes and their roles in various disease states, as well as for accurately assessing biomarkers in exosomes. In addition, extensive clinical sample studies are imperative to validate the practical effectiveness of exosome-based precision assays in the diagnosis and treatment of tumors.

CEA, widely employed as a tumor marker, exhibits overexpression in colorectal, gastric, pancreatic, breast, ovarian, and lung cancers, rendering it instrumental in tumor screening, diagnosis, monitoring, treatment response assessment, and prognostic prediction[37]. During the early stages of tumors, CEA concentration in human serum remains relatively low, prompting a notable interest in the development of an ultrasensitive detection method for CEA based on an aptamer strategy. Huang et al[38] developed an ultrasensitive electrochemical aptasensor based on DNAzyme-assisted amplification, achieving a detection limit of 0.34 fg/mL for CEA. However, there exists a necessity for reinforcing clinical validation studies of aptamer-based CEA assays, particularly concerning real-time quantitative and portable assays[37].

In this study, the top 20 author keywords (Table 5) indicated that the electrochemical method was the most frequently used signal transduction method, with GNPs being the predominant nanomaterial-based signal amplification strategy, and HCR being the most frequently utilized nucleic acid signal amplification technique. However, given the rapid evolution of technologies, this conclusion may be time-sensitive. New signal amplification technologies may emerge in the future.

Electrochemical biosensing is a low-cost, easy-to-use, minimally affected by a low background signal, highly sensitive, and fast real-time system, thereby garnering considerable interest from researchers[39]. The utilization of aptamer recognition and signal amplification techniques augments the electrochemical signal, thereby bolstering detection sensitivity and specificity[40]. Thus, it exhibits broad application prospects in the field of tumor diagnosis and therapy. Since most medical samples (e.g., blood) are typically opaque, rendering optical detection less feasible, electrochemical biosensors have become increasingly important in the medical field. However, the implementation of electrochemical biosensors in real-world sample analyses mandates a comprehensive evaluation of the effects of various interfering factors on detection outcomes, alongside meticulous validation of the stability and reproducibility of the sensors. In addition, ongoing optimization of electrochemical biosensor performance, including enhancements to response time and reductions in detection thresholds, is imperative to provide more accurate and efficient avenues for tumor diagnosis and treatment.

Nanomaterial-based signal amplification strategies have garnered considerable attention. The utilization of nanomaterials significantly improves the sensitivity and detection capabilities of aptamer sensors, surpassing those devoid of nanomaterials[41]. GNPs, a typical nanomaterial, have emerged as excellent carriers for signal transduction and amplification owing to their high surface area-to-volume ratio, surface activity, as well as unique optical, electronic, and chemical properties. When functionalized with aptamers, GNPs can effectively improve target recognition and detection, thereby facilitating advancements in early tumor diagnosis and precision therapy research. For example, Ahmadzadeh-Raji et al[42] immobilized aptamers onto the surface of GNPs via 11-mercapto-undecanoic acid as a self-assembled monolayer to enhance the sensitivity towards target cells. In another study, Shafiei et al[43] employed reduced graphene oxide, chitosan, and GNPs to fabricate a composite material featuring enhanced conductivity and increased effective body surface area, subsequently incorporating aptamers to devise a tumor cell detection method characterized by heightened sensitivity, specificity, and a broader detection range. However, its practical application remains constrained by factors such as low stability, high cost, and complex preparation processes.

HCR, an enzyme-free signal amplification technique, relies on a DNA hybridization reaction that was first introduced to the aptamer field by Dirks RM and Pierce NA in 2004[44]. The HCR system comprises an input strand (initiator) and two sequence-specific nucleic acid hairpins (amplifiers). Upon introduction of the input strand, it triggers hybridization and the alternate strand replacement of the two hairpins, culminating in the formation of an elongated double strand that carries a multitude of detected signals, thereby achieving signal amplification[45]. This technique is highly specific, sensitive, and programmable, and has been widely employed for various bioanalyses, including tumor-related molecular analysis[45,46]. Therefore, HCR emerges as a promising method for nucleic acid signal amplification, and its integration with nanomaterials and/or nanotechnology holds the potential to further improve the detection performance of sensors.

The citation burst of keywords can serve as an indicator of prevailing research trajectories and hotspots. The keywords exhibiting the most robust bursts in the past 3 years were "carcinoembryonic antigen", "electrochemical biosensor", and "nanosheets" (Figure 6B), indicating that CEA and electrochemical biosensors will remain the focus of future research endeavors, alongside the burgeoning interest in nanosheets among researchers. Taken together with the burst in keywords, a trend emerges wherein researchers have shifted their focus from GNPs (2010-2016) to nanosheets (2020-2023). Nanosheets, defined as two-dimensional nanomaterials, have garnered considerable interest from researchers owing to their greater body surface area ratio and superior biomedical properties compared to nanoparticles[47]. In the domain of tumor detection, common types of nanosheets employed for the development of aptamer sensors include carbon-based nanosheets (e.g., graphene oxide)[48], and semiconductor nanosheets (e.g., black phosphorus)[49], as well as metal (e.g., MOF)[50] and transition metal nanosheets (e.g., Ti3C2 MXene)[51].

In signal amplification for aptamer-based tumor detection, the primary role of nanosheets is to serve as a building block for the sensing platform and as a medium for signal amplification. By loading aptamers, nanosheets can capture target molecules, thereby inducing significant signal changes. Such signal changes can be translated into specific electrochemical or optical signals, leading to highly sensitive and specific detection, with the potential for achieving early and accurate tumor detection. However, despite some progress in the preparation of nanosheets, their stability and consistency of structural/compositional parameters remain to be further optimized, and their biosafety and biocompatibility warrant evaluation to ensure their safety and efficacy for in vivo applications[47].

After an in-depth analysis of the keywords, we affirmed that among the signal amplification methods employed in aptamer-based tumor detection, nanomaterial-based signal amplification is the most widely utilized method, with GNPs being the most frequently employed. However, as technological advancements evolve, nanosheets are expected to gradually replace GNPs from their central position in this field. In addition to nanoparticles, other common signal amplification methods include HCR, DNAzyme-based signal amplification, rolling circle amplification, and catalytic hairpin assembly. HCR has emerged as the most popular nucleic acid signal amplification technique owing to its enzyme-free and isothermal characteristics. In practical implementation, each signal amplification technology has its advantages; therefore, the selection of appropriate signal amplification techniques for aptamer-based tumor detection, requires careful consideration of the specific application requirements and conditions.

In addition, the most effective signal amplification methods may encompass a synthesis of multiple advantages. For instance, the concurrent utilization of nanomaterials alongside signal amplification technologies has shown promise in enhancing the detection capabilities of aptasensors. Xie et al[52] developed an ultrasensitive aptamer sensor by coupling MOF signal tags with HCR, resulting in a lower detection limit of 1.34 fM for Mucin 1 by exploiting the unique properties of black phosphorus, GNPs, Co-MOF, and HCR. The combined use of biomaterials and biological techniques for signal amplification in aptamer-based sensor detection presents a viable strategy for augmenting the sensitivity, specificity, and stability of the assay. Moreover, it holds promise as a pivotal approach towards achieving early and precise tumor detection. Realizing this potential necessitates intensified interdisciplinary collaboration and the investment of resources in the future.

Our study exhibits several limitations. Firstly, we exclusively collected articles from the WoSCC database, potentially omitting some publications, even though this database exhibits good quality and comprehensive data commonly employed for bibliometric analyses. Secondly, our study excluded certain types of publications, such as early access and non-English articles, which may have led to oversight of some literature. However, this exclusionary approach aligns with prevailing methods in literature screening. Finally, given the time-dependent nature of publication impact analysis, there is a possibility that newly published high-quality papers were overlooked. However, these limitations exert a relatively minimal impact on the overarching findings and are unlikely to alter the primary trends delineated in this study. This assertion is substantiated by the study's focus on papers published in the last 7 years (80% of the analyzed publications). Furthermore, temporal metrics, including timeline graphs and citation burst analysis, were employed to discern emerging research priorities.

In summary, we performed a bibliometric analysis to elucidate the prevailing trends and frontier areas in signal amplification for aptamer-based tumor detection for the first time. In this field, China and Nanjing University (China) emerge as the leading countries and institutions, respectively, in terms of the number of publications. The most influential researcher and journal were identified as Hasanzadeh M (Iran) and "Biosensors and Bioelectronics", respectively. Currently, the most commonly employed signal transduction method, nanomaterial-based signal amplification strategy, and signal amplification technique are the electrochemical method, GNPs, and HCR, respectively, with exosomes and CEA being the most extensively studied tumor-related targets. Over the past 3 years, research hotspots have focused on exosomes, CEA, electrochemical biosensors, and nanosheets. We believe that two-dimensional nanosheets will be widely employed to develop signal amplification techniques for aptamer biosensors and that the integration of nanocomposites with biotechnology could be a prospective avenue for research exploration in this field.

CONCLUSION

This study represents the first bibliometric analysis of the literature concerning signal amplification in aptamer-based tumor detection. It elucidates the current status, research hotspots, and future research directions in this field, thereby furnishing a crucial reference for researchers.

ACKNOWLEDGEMENTS

We thank the members of the First Affiliated Hospital of Nanchang University for their contribution to the discussions. We apologize to the scientists whose work could not be cited owing to space limitations.