Multiplexed immune checkpoint biosensor (MICB) development is progressing rapidly due to their high specificity and selectivity [3]. Multiplex biosensors require small volumes of sample to diagnose the disease and work simultaneously to detect multiple immune checkpoints. The parallel detection device is growing as it can be potentially translatable for routine clinical applications. However, with these sensors, the analyte tends to take longer to bind with the cell surface without the use of nanofluidic mixing. Hence, these sensors are time consuming and generate a response slowly. For example, a droplet loaded in the electrode can detect the PD-L1 marker and can run multiple samples at once (28 for the multiplexed biosensor on the PD-L1 marker) [42]. Advanced versions of multiplexed electrohydrodynamic biosensors have shown improved performance for biomarker detection [89,90]. However, many of the multiplexed lateral flow nanosensors display this limitation. Additionally, getting and visualizing the target alone for these sensors is also difficult as there are many non-specific interferences displayed in the system. Based on the applications and sample results, biosensors can be broadly classified into two types: (a) high sensitivity and specificity at the cost of a complex system (SPR and multiplexed biosensors) and (b) easy to use and cost and time effective, but not possessing high sensitivity and specificity (colorimetric and EC biosensors).

Another MICB developed by Wuethrich et al. detects soluble PD-1, PD-L1, and LAG-3 in parallel in liquid biopsies, using as little as a single sample drop of approximately 20 µL volume per target immune checkpoint [42]. MICB utilizes a microfluidic sandwich immunoassay with high-affinity yeast cell-derived single chain variable fragments (ScFvs) as well as alternating current electrohydrodynamic in-situ nanofluidic mixing. The high-affinity yeast cell-derived ScFvs are an alternative to the use of monoclonal antibodies and polyclonal antibodies, which are costly and have a limited shelf life. High-affinity yeast cell-derived ScFvs provide longer shelf life without sacrificing specificity and can be mass-produced for a lower cost. The nanofluidic mixing stimulates immune checkpoint interactions with the biosensor while diminishing non-target sensor binding, providing greater specificity for immune checkpoint capture.

MICB has ability to analyze 28 samples in less than two hours. To understand the ability for MICB to detect soluble PD-1, PD-L1, and LAG-3 in liquid biopsies, various concentrations of immune checkpoints were spiked into diluted human serum. While the assay was able to detect a differentiable signal response from the samples with the immune checkpoints, fortified samples had a concentration equal to the prognostic cut-off, which may differ from the threshold for cancer detection [42].

Exosomes are the key players in communication at the intercellular level through surface biomarkers. For instance, exosomal proteins are fundamental in cancer development, drug resistance, and differentiation etc [91]. Expression of PD-L1 in exosomes is a biomarker that is overly expressed in lung tumors. Hence, many exosomal proteins that include exosomal immune markers like PD-L1, can be promising cancer biomarkers. In a recent study, researchers constructed a compact surface plasmon resonance biosensor that detects exosomal PD-L1 and EGFR [92]. Looking at the different serum samples of patients with lung cancer, researchers used this biosensor to determine the specificity and sensitivity of the biomarker in these samples.

This biosensor worked through optical resolution using laser beams. Essentially, two laser beams were used in which one allowed for the SPR to interact with the exosomal proteins on the surface of the sensor. The other beam was used as reference, and the two photodetectors quantified the exosomal proteins by magnifying the intensity of the initial beam and the reflection beam. Overall, in terms of results, the SPR biosensor was much more effective in detection of exosomal PD-L1 in comparison to conventional methods like ELISA [93]. First and foremost, the study depicts that the SPR biosensor was very accurate and effective in detecting the PD-L1 in serum samples including that of a Stage III lung cancer patient; however, the method of ELISA was unable to detect PD-L1 in general. These serum samples were all 50 microliters. Additionally, through these biosensors, researchers proposed that the exosomal PD-L1 can be a biomarker (although this still needs to be confirmed through larger sample sets) for lung cancer diagnosis as the SPR biosensor detected much more exosomal PD-L1 in lung cancer patients compared to the normal controls [92]. The sensitivity and specificity of the SPR biosensor was much greater than the ELISA testing as the ELISA test could not detect any of the PD-L1 in serum samples in which there was a very low abundance of the exosomal PD-L1.

Biosensors could play a pivotal role in augmenting cancer immunotherapy for treatment monitoring and evaluation of disease condition. For example, several study results revealed that the nano biosensor can be utilized cytokine secretion from individual cells for hours [94]. A specific biosensor is combined with a gold nanohole array sensor and a microfluidic-based system for accurate depiction and detection of cytokine secretion within a cell-to-cell basis. Additionally, some groups have worked on detection of the IL-2, IFN-γ, and TNF-α cytokines using gold nanorod-based biosensors [95]. Using these different types of sensors, researchers showed that immunoanalysis could be done on a smaller volume of analyte and a shorter timeframe than conventional ELISA methods [96]. Thus, these nanomaterial-based biosensors can be employed for more versatile applications than detection of a major immune checkpoint marker such as PD-L1.

Clustered regularly interspaced short palindromic repeats (CRISPR/Cas) based biosensors have also become more important for rapid detection of nucleic acids, exosomes, tumor DNAs, etc. CRISPR/Cas sensors use Cas effectors to cleave a specific DNA sequence. One of the main effectors, Cas9, has become well known due to its effective ability of editing genomes, identifying nucleic acids, and regulating transcription [97,98]. Specifically, Cas9 uses single guide RNA to help target a specific double-stranded DNA and cleave it for the purpose of selective genome editing [97,98]. In addition, the usage of nuclease-deactivated Cas9 can allow for binding and rebinding to the targeted double-stranded DNA, which is essential as fluorescent enzymes can be fused to the DNA to allow for detection of specific nucleic acid sequences due to the fluorescence and light emission [83,84]. This fusion of integrating Cas9 with a fluorescent enzyme was instrumental for researchers to detect DNA sequences of the American Zika virus. Other types of Cas effectors such as Cas12 and Cas13 have also been explored. Wherein, Cas12 effector is extremely important due to its ability to detect single-stranded DNA, which adds more to the detection of the different nucleic acids rather than focusing only double-stranded DNA with Cas9 effector. However, the main reason researchers have started to focus on CRISPR/Cas-based biosensors for cancer detection is due to the Cas13a effector. This effector works with the collateral cleavage activity of single-stranded RNAs. When there is a specific RNA of interest, the Cas13a effector cleaves RNA, the collateral cleavage of the reporter RNA (reRNA) which allows for the detection of the RNA with accurate quantification [85]. This effector is key for recognition of many miRNAs which can be either up-regulated or down-regulated. Hence, by being able to detect levels of miRNAs rapidly and effectively, this field of CRISPR/Cas-based biosensing is becoming a hot topic and displays promise for the early detection of cancer. However, one key limitation is that these biosensors involve sophisticated systems to run a tedious process to function fully [97,98]. Hence, reading these biosensors and their detection of the specific analyte requires well-trained professionals to understand the process and readout. Furthermore, the sensitivity of the biosensors is relatively low when compared to the other amplification methods [97,98]. Though many researchers are working on CRISPR/Cas-based biosensors, none of them could meet the needs of in vivo testing due to low sensitivity detection. Despite these issues, there is potential for the development of biosensor based on the Cas13a effector and the collateral cleavage activity detection [99].

Globally, the market size and demand for biosensors is rapidly increasing [98]. In 2026, the market size is expected to expand to about 37 billion dollars for all the different types of biosensors (electrochemical, FRET, piezoelectric, optical, etc.). However, improvements in biosensors are still a challenge because of the low concentration of biomarkers expressed over the cells. Nanomaterials-based detectors have the potential to amplify the signals with limit of detection at pico- or femtomolar concentration. In addition, the COVID-19 pandemic in 2020 has shown us the potential need of biosensors. Hence, in this review we have outlined current improvements and strategies in biosensors over the last decade for biomedical applications have been explored.

The detection of immune checkpoints via liquid biopsy presents an attractive option for early cancer detection. Currently, immune checkpoints are not widely used for diagnostics. Instead, they are utilized as prognostic markers for cancer and as predictors for the success of various immunotherapies. The biosensors proposed in recent years from the studies of Xu et al. [25] showcase the various advantages of using these biosensors for detecting small concentrations of PD-L1. For the quantification of different immune checkpoint markers like PD-L1, the studies showcased the limitations of ELISA and immunohistochemistry. For example, with ELISA, it is difficult to detect PD-L1 with very small concentrations. However, PD-L1 in multiple types of serum media is found in very small concentrations and thus, ELISA becomes extremely inaccurate in many situations. Immunohistochemistry is also an employed method that becomes very time-consuming and has very complex procedures. With these surface plasmon resonance-based biosensors and the multiplex biosensor, the studies show the simple-to-use nature of the biosensors, the very high specificity and sensitivity of the sensors, as well as their time and cost effectiveness. One way to ensure cost-effectiveness is a sensor that uses very cheap single-chain variable fragments from yeast cell lines in replacements of the very costly antibodies of the different immune checkpoint proteins that are used in the conventional methods.

Biosensors have been developed to detect the concentrations of the immune checkpoint markers in serum samples e.g., PD-L1 [42], but other potential IC markers can be identified through same approach. These biosensors demonstrated a few unique features with fine differences in the benefits and limitations of using one over the other. Table 1 list the various advantages and disadvantages of these biosensors that are reported in the relevant literature [31,42,83,84,90]. Developments in surface plasmon resonance (SPR)-based biosensors development, especially for the detection of immune checkpoint markers in early detection of cancer, have progressed well due to several key advantages [31,92].

LSPR biosensors show very high specificity and sensitivity (LOD: 1–5 pg/mL) for the immune checkpoint marker PD-L1 [31]. Furthermore, these biosensors are highly selective for the marker in comparison to the normal detection methods, e.g., immunohistochemistry, and can be integrated with a label-free detection system. Most of these sensors use fluorescent labeling or some other type of labeling approach to analyze the different analytes. However, the detection of analytes was required with different types of media which resulted in non-specific binding and high background signals. In addition, receptor labeling requires extensive time, effort, and optimization for the development of accurate biosensors for reproducible results. These limitations do limit the biosensors’ usability. However, there is no additional labeling step required since this biosensor relies on physical properties of the analyte immobilized on a surface. Hence, these biosensors do not require nearly as much labor nor time to work effectively. Additionally, the biosensors allow for real time detection of the analyte as the analyte is localized into a compact surface; thus, there is no alteration of binding sites which could possibly occur with biosensors that require a labeling system.

SPR-based devices can cause configurational changes of the marker or analyte being detected by biosensors [32]. These biosensors use a mechanism of action where the analyte gets immobilized on the surface, often causing lower binding affinities due to the configurational changes of the immobilized receptor which, can cause the analyte binding ability and make it hard to detect the analyte accurately. SPR biosensors detect the marker or analyte by a change in wavelength with the aid of gold nanomaterials. Using these nanomaterials and immobilizing the analyte is a complex system to use for the commercialization of the product on a large scale.