Integration of microfluidic chips with biosensors

Abstract

The potential threat posed by disease outbreaks to diagnostic instruments demands the development of more effective biosensor technologies to counteract the risks. Diseases like SARS-CoV-2, Ebola, malaria, cholera, and many more have demonstrated beyond the limits of health care that new advancements are required for early detection and diagnosis. The rising number of diseases outbreaks has led to an increasing demand for biosensors that are more effective and quicker to utilize in healthcare settings. A biosensor incorporated with microfluidic chips offers an improved detection compared to traditional or classical biosensors. Microfluidic chips improve the performance of the biosensors by allowing automation, mixing, separation, throughput processing, and transport of the analytes to desired reactors. A biosensor incorporated with a microfluidic chip has improved sensitivity, easy operation and can use small volumes of samples to process the results. The effectiveness of biosensors depends also on the materials used in its fabrication and there are many materials used for fabrication which are reviewed in this work. This paper reviews the potential advantages of the use of microfluidic chips to enhance the performance of biosensors, materials used to fabricate the chips, and potential electrodes incorporated into microfluidic chips which improve the detection time by shortening the processing time for biosensors at the point of care service. This work also reviews new technologies which are not previously addressed other reviews including, integration of cell-imprinted polymers with microfluidic sensors and delved into future technologies outlook.

Article Highlights

• Highlight 1: This work reviews biosensors integrated with microfluidic chips.

• Highlight 2: The work reviews different materials used to fabricate microfluidic chips and their impact on biosensing.

• Highlight 3: The works also highlight the integration of electrodes into microfluidic chips to improve biosensor performance.

1 Introduction

The ability of biosensors to identify infections quickly and widely has been put to the test by outbreaks of SARS-Cov-2, Ebola, malaria, cholera, HIV, and tuberculosis. Biosensors such as optical, enzymatic, immunosensors, etc. have been proven to be effective in performing detection, yet some require large volumes of samples, and some require trained personnel for operation [1]. The rise in disease outbreaks demonstrated the need to develop biosensing equipment, specifically to be used more efficiently at point-of-care settings for rapid and mass detection [2]. A biosensor is an analytical tool that is used in different applications, which include drug discovery, medical diagnostics, defense and security, food safety, environmental monitoring, and agriculture [3]. Potential biosensors can aid in the early identification and diagnosis of diseases, as well as monitoring progress after detection [4]. Biosensors can also detect pollution in water, and disease-causing germs in biological fluids such as saliva, tears, blood, urine, and perspiration. [5]. Using a microfluidic chip, which is a collection of microchannels used to control fluids from micro to nanosized, can improve the performance of the biosensor [6, 7]. Microfluidics is a multidisciplinary field that encompasses engineering, physics, microtechnology, chemistry, biochemistry, nanotechnology, and biotechnology [5]. The integrated microfluidic chip provides the biosensor with the advantage of small analyte volumes, automation, and multiplexity. The use of microfluidic chips will enhance biosensor performance at the point of care by boosting sensitivity, selectivity, and quick detection [8]. Microfluidic chips can be integrated into any kind of biosensor, including optical biosensors, which use the chip to analyze molecular interactions with light in real-time, and microarray biosensors, which use the chip to channel fluids to microwells for molecular analysis, and other types of biosensors that will be covered in later sections [9]. Other alternative means of improving the performance of biosensors apart from microfluidics include using nanoparticles [10], machine learning [11,12,13,14] and quantum technologies [15,16,17,18,19].

In this work we also review the materials used to build microfluidic chips. Microfluidic chips can be made from a variety of materials, and each material has an impact on the chip's mechanical strength, thermal stability, and biocompatibility. As of late, PDMS has been suggested as the material of choice for most chips that are manufactured. This recommendation is predicated on PDMS's lower cost, biocompatibility, and optical transparency [20]. Traditional materials that are also suggested to be used for the optimal manufacture of microfluidic chips include silicon, glass, ceramics, and various plastics (Fig. 1) [21]. Traditionally, several techniques are used to create microfluidic chips; these techniques mostly depend on the materials utilized; for example, certain techniques may not be applicable to plastic. One of the most advised techniques for creating the microfluidic chip is photolithography, yet there are now numerous approaches under consideration, including etching and 3D printing. [9, 22, 23].

Fig. 1

Materials used to fabrication microfluidic chips for various applications in biosensing microfluidic applications [30]

We also review applications of microfluidic chips in this work. Biological cells can be cultured, seen, and tested using microfluidics, and the addition of electrodes to microfluidic channels increases the systems’ functionality, sensing, and testing capacities [24]. An electrode is a solid electric conductor that transports electric current into nonmetallic materials, liquids, gases, plasmas, or vacuums [25]. In the past, platinum (Pt) electrodes have been thought to be the most stable when compared to electrodes constructed of gold (Au), silver (Ag), cobalt (Co), copper (Cu), etc. [26, 27]. Another significant aspect in microfluidic chips is microfluidic platforms, which are a set of devices that allow operations to be carried out in the microfluidic chip [28]. The microfluidic platforms include capillary, auxiliary, etc. Platforms can enable fluidic activities including separation, extraction, and transportation. [29].

Microfluidic chip platforms also help in recovering, quantifying, and analyzing samples and this feature has made the microfluidic chips to be used in diverse areas of science [31]. Additionally, microfluidic chip platforms facilitate sample recovery, quantification, and analysis; this feature has led to the widespread application of microfluidic chips in several scientific fields [32, 33].

2 Overview of microfluidic chip-based biosensor systems

When microfluidics chips are integrated into biosensors, they become a viable substitute for traditional laboratory analysis. Microfluidic chip integration is carried out on a traditional biosensor. The cost of operation and assay times are significantly lower with microfluidic-based biosensors than with traditional biosensors, and at low detection capacities, specificity increases [34]. Traditional microfluidic biosensors consume little analytes, which results in high precision; the use of a microfluidic chip standardizes the improvements [35]. Furthermore, traditional biosensors require a large amount of volume to attain equilibrium, which results in a poor diffusion rate and a prolonged response time, poor signal and noise levels are further issues brought on by this. Moreover, microfluidic biosensors provide improved signal quality, reduced noise, and quick response times because of the small volume use [36]. Microsystems such as microchannels and microchambers can perform comprehensive analyses, such as continuous sampling and sample treatment (separation and mixing, or pre-concentration), because they are small [37]. Biosensors are widely used in point-of-care diagnostics, and their combination with microfluidics has enhanced the diagnostic uses. Enzymatic, immunosensor, optofluidic, and microarray biosensors are a few of the microfluidics-based biosensors that are more effective in diagnosis (Table 1) [38]. This section delves into a detailed discussion of microfluidic-based biosensors, highlighting the significance of microfluidic chips.

Table 1 Different types of microfluidics biosensors with their application, advantages, and disadvantages at point of care application

2.1 Overview of microfluidic-based biorecognition biosensors

2.1.1 Integration of the microfluidic chip onto enzyme immobilized biosensor

Enzymes are common biocatalysts that improve the rate of biological reactions. To detect the target analyte, an enzyme-immobilized microfluidic biosensor needs enzyme’s capacity to bind and catalyze reactions [39]. The process of identifying analytes involves multiple processes, which are contingent upon the subsequent factors.: (i) the analyte is metabolized by the enzyme and the enzyme concentration is roughly calculated by measuring the catalytic change of the analyte by the enzyme; (ii) because the analyte inhibits or activates the enzyme, the concentration of analyte is related to decreasing enzyme product formation; and (iii) tracking of the change in enzyme characteristics [40, 41]. Enzymatic immobilized microfluidic biosensors provide a strong platform for point-of-care diagnostics. These biosensors are effective in detection because of the enhanced sensitivity because of microfluidic chip integration [42]. Enzymes are immobilized onto an appropriate transducer in an enzymatic immobilized microfluidic biosensor, which reacts with a specific analyte to produce a specific signal [43]. An aptamer is a sequence of single-strand oligonucleotides (DNA or RNA) with a variable region made up of tens of nucleotide bases. [44]. These random sequences provide each aptamer with a particular three-dimensional shape and possible binding capabilities to target molecules such as chemical substances metal ions, and proteins (circulating protein, membrane protein) [45]. Figure 2 shows an example of an enzyme-based microfluidic biosensor; (a) labile biological components, indicating the important analyte that the enzyme needs to metabolize [46], (b) An electrical signal is produced when analytes interact with a microfabricated device in bio-recognition, illuminating the detection process. The signal is produced as a result of the analyte blocking or activating the enzyme [47], (c) Enzyme immobilization in a fully packaged microfluidic channel illustrates the immobilization procedure with the essential components necessary for the biosensor to function [48]. The microfluidic chip acts as the part that holds the transducer and microreactors, and channels assist in sample mixing, separation, and flow to directive chambers [49, 50].

Fig. 2

The image depicts a schematic of a microfluidic biosensor device, illustrating different stages and components involved in biosensing using microfluidic channels and electrochemical detection [51]

2.1.2 Integration of microfluidic devices onto immunosensor

Solid-state devices called immunosensors link a transducer and the immunochemical response [52]. They are among the most important classes of affinity biosensors since they produce stable complexes, like those produced by immunoassays, by the molecular recognition of antigen-specific antibodies [53]. The immunological complex can be detected and quantified label-free using modern transducer technology, which differs from immunoassay [54]. The immune system is extremely capable of distinguishing between self and non-self. As part of the immune defense system, the body synthesizes antibodies (Abs) with great specificity to recognize foreign species which are antigens (Ags). The immune system of organisms can detect the presence of Ags and respond quickly by producing Abs with high binding affinity. Immunosensors are based on the immunochemical reactions of antigens and antibodies [55]. To identify the antigen in the sample, the antibody frequently interacts with a transducer surface as a recognition element. The most popular technique for detection is called enzyme-linked immunosorbent assay (ELISA) [56]. For instance, using the Cu2 + -OPD reaction system, Binfeng et al. created an automated microfluidic device specifically built for the dual-mode detection of E. coli in real samples (Scheme 1). In short, the automated gadget that was built combines a computerized control system with a microfluidic chip that resembles a seashell. The control button on the screen of the automated equipment can be used to direct the mixing of samples in SMC. Cu2 + can oxidize OPD in the Cu2 + -OPD reaction system to produce OPDox, which can fluoresce and has a yellow tint. Cu2 + can be converted to Cu + by E. coli, which can then stop OPD from oxidizing. Notably, the colorimetric and fluorescence of OPDox may be observed with the naked eye, and this change only takes place when live bacteria are present to carry out the reaction process [57]. Figure 3 illustrates how the immunochemical reactions of antigens and antibodies depends on surface geometry, which can be either three-dimensional (3D) or planar (2D). Figure 3a, b, and c shows immobilization of the receptors on 2D microfluidic surface coated with various substrates such as gold, platinum or graphene. Figure 3d, e, and f depicts the immobilization of receptors on 3D surfaces. Receptors can be added to 3D structures to functionalize them once they have developed on a substrate or after assembling a device's component parts. Here, the same technique used by the microfluidic chip in microfluidic-based enzyme biosensors is the same in microfluidic-based immunological biosensors, microfluidics still plays the same role.

Scheme 1

A microfluidic device that is automated and downsized, utilizing adual-mode readout mechanism, designed to detect E. coli

Fig. 3

Techniques for immobilization on 2D surfaces and 3D geometries for frequently used microfluidics receptors, such as protein (a) immobilization on glass or silicon, b metal, c polymer or PDMS, respectively, d immobilization of beads arranged in a three-dimensional configuration. Materials for beads include ferromagnetic materials, polystyrene, silica agarose, e hydrogel immobilization, PEGDA, agarose, polyacrylamide gel, and chitosan are examples of hydrogels that are frequently utilized, f immobilization by membrane porosity. For instance, porous silicon and polymer monoliths (such as ethylene dimethacrylate, acrylamide, and 2-hydroxyethyl methacrylate) can be used to create porous membranes. As an alternative, porous membranes like paper, nitrocellulose, polyvinylidene fluoride, and polycarbonate are also frequently utilized [58]

2.1.3 Imprinting of microarray on microfluidic devices

Microarrays are a useful technique used in molecular biology that allows for the high throughput and multiplexed detection of biological analytes. They are made up of a variety of biological components that allow many tests to run at once. Researchers frequently use biomolecules (such as DNA and protein) and cellular microarrays to detect mutations, drug discovery, identification, and classification of tumors, profiling of gene expression, etc. [59]. DNA microarray is a widely used technique to measure gene expression levels. It is made up of numerous small patches that interact with the genetic material of a sample to form a double DNA strand [60]. For instance, a colorimetric vertical-flow DNA microarray for Neisseria meningitidis detection was created. This technique leverages the screening capabilities of DNA microarrays in a paper format in addition to isothermal amplification using recombinase polymerase amplification. Additionally, Ma et al. created a DNA microarray technique that allows pneumonia patients to simultaneously identify fifteen different bacterial species from their respiratory tract; 16S rRNA genes and other particular genes of each pathogen were the assay's target, and 103 copies/μL was the detection limit [61]. As illustrated in Fig. 4a, a simple microfluidic device with six parallel channels was devised and manufactured with flow-through reaction cells. Photonic crystal beads were injected and trapped in a metal microchannel array [62]. In application, the sample interacts with probe molecules that are immobilized on the surface of the array of photonic crystal beads as it passes through the metal microchannels [63]. By transferring samples through microarrays, microfluidic technology can improve exposure. Additionally, printing innovative low-density and high-density arrays is a great use for microfluidic chips [64]. The results of the multiplex assay are displayed in Fig. 4b and c, where an epifluorescence and an epi-white light image are taken from the bottom of the reaction cell as the detection and encoding images, respectively.

Fig. 4

Photonic crystal beads arrangement in a microfluidic chip and image capture. a The reaction cell’s three-dimensional illustration and flow through microfluidic chip. b To obtain the ultimate assay result, an epi-white light and an epi-fluorescence image are obtained from the bottom of the reaction cell’s microchannels as detection and encoding images, respectively. c The schematic layout of an inverted optical microscopic detecting system home built [62, 65]

2.2 Overview of microfluidic chip-based biosensing techniques

2.2.1 Integration of microfluidic devices onto optical biosensors

An analytical device that has a biorecognition sensing element and an optical transducer approach is known as an optical biosensor [66]. The key purpose of an optical biosensor is to give a signal proportionate to the concentration of the sample under investigation. Among other biological substances, optical biosensors may identify enzymes, antigens, antibodies, receptors, whole cells, nucleic acids, and tissues [67]. There are various optical biosensors which include, surface plasmons resonance, localized surface plasmons resonance, photonic crystal optical biosensors (see Table 1), mentioned a few of them. The optical field and a biorecognition component work together to provide optical detection, which can be either label-free or label-based optical biosensing [68]. Optical biosensors offer major advantages over standard analytical methods since they can detect numerous biological and chemical components directly, in real-time, and without the usage of labels [69]. In summary, the measured signal in the label-free mode is created directly by the interaction of the substance being examined with the transducer [70]. Label-based sensing, on the other hand, employs a label followed by a fluorescent, colorimetric, or luminescent technique to generate the optical signal [71]. Optical sensor integration with microfluidic chips involves the integration of light-based detection techniques with microfluidic devices to perform real-time monitoring of microfluidic reactions. A schematic of the sensor-integrated microfluidic chip is shown at the top in Fig. 5, The photonic crystal biosensor and the flow channels are self-aligned on a single flexible plastic sheet [72]. The combination of these technologies allows precise control and manipulation of small fluid volumes and enables the analysis of various physical and chemical properties of these fluids [73].

Fig. 5

Diagram illustrating how the high-resolution imaging detection device is integrated into the photonic crystal biosensor integrated microfluidic assay chip [72, 74]

2.2.2 Integration of microfluidic devices onto electrochemical biosensor

Electrochemical biosensors provide great sensitivity, fast response times, and inexpensive costs by using biorecognition elements to generate signals such as current or potential. For short-term uses mostly, they evaluate target concentrations directly [75]. For reactions to be optimized, electrode materials are essential, particularly novel nanomaterials with superior electrical characteristics and biocompatibility. Microfluidics are utilized to confine the electrode in an electrochemical biosensor since the electrode’s biochemical reaction with the target analyte during biorecognition is what produces the electrical signal [76]. The electrodes function as transducers, converting the biological signal into electrical impulses, and are embedded within the microfluidic chip. The three main ways that electrical signals are produced are: (1) through current resulting from the electrochemical oxidation or reduction of the analytes on the electrode surface; (2) through current resulting from voltage generated by the analytes’ behavior; and (3) as a function of frequency as a result of the interaction between the analyte and electrode surface [77]. Figure 6 illustrates microfluidic electrochemical biosensor, (a) microfluidic structure that permits multiple analyte inlets and outlets, reaction chamber that conducts the biological reaction, and air valve, also known as pneumatic control lines, which regulates fluid flow. The electrodes are integrated into the bottom layer of the biosensor [78], (b) a microfluidic biosensor system consisting of an electrode, data processing and analysis software, and a sensor PC integrated with the sensor chip. Au and Ag electrodes are the electrodes utilized in this biosensor. Because microfluidic chips require small sample volumes, they improve sensitivity, speed of analysis, and selectivity [79].

Fig. 6

Schematic illustration of the microfluidic system for electrochemical analysis in a single device. a Schematic description of the microfluidic system integrated electrochemical sensor, b the microfluidic electrochemical biosensor is connected to a PC and a control box to enhance transportability [80]

Researchers have also advanced electrochemical biosensors due to the recent surge in nanotechnology research. To quickly and accurately measure pathogens in patient samples at clinically meaningful quantities, a self-assembled monolayer (SAM) based nucleic acid biosensor for bacterial 16S rRNA has been described. Modifying the amperometric biosensing platform to carry out pathogen identification, antimicrobial susceptibility testing, and multiplex detection of protein and nucleic acid biomarkers simultaneously is possible. In microfluidic formats, these biosensors have also been used to provide point-of-care and automated analysis [81].

2.2.3 Integration of microfluidic devices onto piezoelectric biosensor

The primary form of piezoelectric biosensors is a quartz crystal microbalance, which functions as a transducer by binding antibodies, aptamers, particular receptor proteins, etc. to the crystal’s surface. When target analytes pass through coated channels, they attach to biorecognition elements like aptamers or antibodies. This binding is indicated by a change in the mass or mechanical property of the quartz [82]. The piezoelectric materials coated inside the surface of microfluidic channels allow the conversion of surface mass change into resonance frequency change, which is then used to detect MBs [82]. The piezoelectric microfluidic biosensors can also be employed for real-time analysis by monitoring the change in target analyte concentration where the electric signal is a result of the detecting concentration [83] Piezoelectric microfluidic biosensors are advantageous because they use small volumes and offer fast detection, sensitivity, and multiplex analysis capabilities. Figure 7 depicts the piezoelectric biosensor with elements such as a gold electrode, oscillator, and piezoelectric crystal. In piezoelectric biosensors, an applied current or potential causes a crystal to deform elastically. The alternating electric field at a specific frequency produces a wave in the crystal. On the surface of the crystal, where the biorecognition element is coated, the analyte is either absorbed or desorbed, the resonant frequency shifts, indicating the possibility of binding [100]. Yang et al. demonstrated the use of a lead titanate zirconate (PZT) ceramic resonator as the transducer in a piezoelectric biosensor for the label-free identification of cancer biomarkers.

Fig. 7

Schematic illustration of microfluidic piezoelectric biosensor. The biosensor is made of piezoelectric crystal with Au electrodes. The Au electrodes are coated with protein layer to which antibodies are incorporated. The biosensor measures the frequency as the results of analytes binding to the antibodies. The frequency is measured by frequency counter and detected by oscillator; the frequency measured is used to provide quantitative analysis of the analyte concentration

2.3 Advances in molecularly imprinted polymers (MIPs) in microfluidics

Molecular imprinting is a technique used to create template-shaped cavities in polymer matrices with memory of the template molecules to be targeted [91]. This process essentially forms a “molecular memory” within a material, allowing for the selective recognition and binding of specific molecules. The integration of cell-imprinted polymers (CIPs) with microfluidic sensors represents a significant and emerging trend in the fields of electrochemical and fluorescent biosensing. This approach combines the high selectivity of molecular imprinting technology with the versatility and efficiency of microfluidic devices, paving the way for advanced biosensing applications. This section discusses recent advancements in this area and addresses the gap in literature by highlighting key studies.

Advancements in Molecularly Imprinted Polymer (MIP)-Based Biosensors.

• 1. Point-of-Care Devices: The integration of molecularly imprinted polymers in point-of-care devices has been particularly impactful. Park et al. [92] discusses the recent advances in designing biosensors that are integrated into point-of-care devices, focusing on biomolecule sensing and intraoral fluid testing. These developments have potential to significantly enhance the practicality and applicability of biosensors in healthcare settings. The work by Park et al. [92] focused on the development of MIP-based biosensors integrated into POCT devices for intraoral fluid testing. Their approach involved synthesizing MIPs with high selectivity and chemical affinity for specific biomolecules, thus improving diagnostic accuracy and stability in harsh environments. The paper [92] highlights several practical applications of MIP-based biosensors, including disposable POCT devices for monitoring biomarkers in various biofluids. The authors also discuss the potential for wearable MIP-based biosensors for continuous health monitoring, particularly for oral diseases like periodontitis. Future research directions include improving the selectivity, stability, and scalability of MIP-based biosensors for broader clinical and environmental applications. A key limitation highlighted for the work is a potential challenge in mass production and scalability. This technology paves the way for the development of reliable, non-invasive diagnostic tools in healthcare. The main highlights of the work done in [92] include

  • Enhancement of Diagnostic Capabilities: MIPs improve the analytical performance of POCT devices by providing high selectivity and stability.

  • Versatility in Applications: MIP-based biosensors are suitable for detecting various biomarkers in different biofluids, applicable in healthcare and environmental monitoring.

  • Future Potential: The paper identifies wearable MIP-based biosensors as a promising area for continuous health monitoring, with potential applications in personalized medicine.

• 2. Fluorescent Bacteria Detection: Doostmohammadi et al. [93] describe an innovative approach using CIP coated microparticles in a magnetophoretic microfluidic device for detecting fluorescent bacteria in water. This method demonstrates an important advance in environmental monitoring and public health by enabling rapid and sensitive detection of bacterial contaminants. Doostmohammadi et al. [93] reported that their microfluidic platform could detect biomarkers at extremely low concentrations, making it suitable for early disease detection. The optimized microfluidic design improved fluid handling and sensor response time, essential for real-time diagnostics. The paper [93] introduces a novel method for rapid and low-cost detection of bacteria in water using cell-imprinted polymer coated microparticles (CIP-MPs) integrated into a magnetophoretic microfluidic device (Fig. 8). This approach aims to address the need for efficient point-of-need (PoN) bacterial detection, particularly for pathogens like Escherichia coli (E. coli), which pose significant health risks through contaminated water and food sources.

  Fig. 8

  

  Experimental apparatus and procedure for the fabrication of microfluidic devices. A The fabrication and implementation of a microfluidic device made of PDMS-glass within the 3D-printed fixture that is responsible for maintaining the magnets in their proper position. B An genuine image of the microfluidic device that has been installed in the 3D-printed fixture. The CIP-MPs are observed to accumulate around the oval-shaped soft magnets in the bottom right inset. C Experimental setup that includes the microfluidic-fixture device mounted on a fluorescence microscope and connected to two syringe pumps for the passage of CIP-MPs and bacteria into the channel

  The study utilized fluorescent magnetic CIP-MPs to capture and detect bacteria. The microfluidic device incorporated soft ferromagnetic microstructures to enhance the accumulation of CIP-MPs within the microchannel, optimizing the magnetic field distribution for effective bacteria capturing. The key steps included:

  • 1. Synthesis of CIP-MPs: The CIP-MPs were created using a two-step polymerization process, incorporating polystyrene fluorescent super-paramagnetic microparticles and E. coli OP50 as templates.

  • 2. Microfluidic Device Fabrication: A PDMS-on-glass microfluidic device was designed with integrated ferromagnetic microstructures to trap CIP-MPs. The device was fabricated using standard photolithography and soft lithography techniques.

  • 3. Bacteria Detection: The fluorescence intensity of CIP-MPs was monitored before and after exposure to bacteria suspensions, with changes in fluorescence indicating successful bacteria capture.

2.3.1 Key findings

• 1. Optimization of Magnetic Microstructures: Numerical simulations showed that oval-shaped soft magnets with a low aspect ratio provided the best magnetic field distribution for capturing CIP-MPs.

• 2. Flow Rate Effects: Lower flow rates (e.g., 0.05 mL/min) resulted in higher bacteria capturing efficiency, as slower flow allowed more interactions between bacteria and CIP-MPs.

• 3. Detection Sensitivity: The sensor exhibited a detection limit of 4 × 10² CFU/mL and a dynamic range of 10²–10⁷ CFU/mL. The fluorescence intensity of CIP-MPs decreased significantly upon binding with bacteria, confirming the method's effectiveness.

• 4. Specificity: The device demonstrated some specificity for E. coli over Sarcina bacteria, though further studies are needed to fully validate its selectivity for different bacterial strains.

The developed magnetophoretic microfluidic device effectively captures and detects bacteria using CIP-MPs, offering a promising approach for PoN bacterial detection. This system's advantages include rapid, simple, and versatile bacterial monitoring, with potential applications in water surveillance and environmental monitoring. Future work in this will focus on enhancing the device's portability and selectivity for broader practical use.

• 3. Development of Molecularly Imprinted Biosensors: Wu et al. [94] provide a comprehensive review of the state of the art in the development of molecularly imprinted biosensors. Their discussion covers various sensing mechanisms and highlights the potential of these biosensors to revolutionize the detection and analysis of complex biological samples. The work done in [94] provides a comprehensive review of the development and applications of MIP-based biosensors. These biosensors are advanced analytical devices designed for specific detection of target analytes using MIPs as synthetic receptors. MIPs, often referred to as “plastic antibodies,” offer high selectivity, stability, and cost-effectiveness compared to natural bioreceptors like antibodies and aptamers. MIPs are synthesized by polymerizing functional and cross-linking monomers in the presence of a template molecule. The process involves three main steps:

  • 1. Auto-assembly of monomers with the template.

  • 2. Polymerization to form a highly cross-linked structure.

  • 3. Removal of the template, leaving behind specific binding sites complementary to the target molecule.

MIPs are advantageous due to their high stability, reusability, and low cost, making them suitable for various biosensing applications. The paper discusses strategies to optimize MIP-based biosensors, including the rational design of molecular recognition sites and combining MIPs with biomolecules to enhance selectivity. However, challenges remain, such as the need for refreshable sensors and effective signal amplification in complex environments.

MIP-based biosensors have diverse applications, categorized into several types:

• 1. Electrochemical Biosensors:

  • o These sensors rely on electro-active molecules to generate a detectable signal. MIP-based electrochemical biosensors offer improved sensitivity and stability over traditional methods.

  • o Example: An MIP-coated gate field-effect transistor (FET) for glucose detection demonstrated high selectivity and sensitivity.

• 2. Optical Biosensors:

  • o Optical biosensors include fluorescence sensors, surface plasmon resonance (SPR) sensors, and surface-enhanced Raman scattering (SERS) sensors. They offer high sensitivity and the potential for real-time monitoring.

  • o Example: An MIP-based LSPR sensor for protein detection showed enhanced sensitivity due to the rational selection of functional monomers.

• 3. Color Biosensors:

  • o These sensors provide simple, accurate, and stable detection methods, often based on structural color changes.

  • o Example: An MIP-based structural color contact lens for monitoring drug release demonstrated a color change corresponding to the target analyte concentration.

• 4. Wearable Biosensors:

  • o Wearable MIP-based biosensors are emerging for continuous health monitoring. These sensors need to be flexible, stretchable, biodegradable, and self-healing to integrate seamlessly with the human body.

  • o Example: A wearable MIP-based electrochemical biosensor for lactate monitoring in sweat showed high sensitivity and stability.

The development of MIP-based biosensors has made significant progress, offering advantages in sensitivity, stability, and cost-effectiveness. These biosensors are poised to impact various fields, including environmental monitoring, medical diagnostics, food safety, and anti-terrorism. Future advancements will focus on enhancing the intelligence, mobility, miniaturization, integration, and diversification of these sensors, driven by the rapid development of the Internet of Things and mobile internet technologies. The integration of artificial intelligence and advanced signal processing will further expand the applications and capabilities of MIP-based biosensors.

• 4. Conductometric Detection of Bacteria: Akhtarian et al. [95] focus on a microfluidic sensor that employs cell-imprinted polymer-coated microwires for the conductometric detection of bacteria in water. This study illustrates the effectiveness of CIPs in enhancing the sensitivity and selectivity of microfluidic devices for environmental and health applications. The paper [95] presents a novel, low-cost, and portable microfluidic sensor for the rapid and on-site detection of bacterial contaminants in water. The sensor utilizes CIPs coated on microwires to selectively capture bacteria, specifically E. coli. This technology addresses the need for quick, inexpensive, and accurate water quality monitoring.

  • 1. Materials and Preparation:

    • o The sensor employs stainless steel microwires (SS-MWs) coated with CIPs, designed to capture E. coli bacteria.

    • o The CIPs are synthesized using a polymerization process involving functional monomers and a template molecule (E. coli). This process creates specific binding sites that mimic natural antibodies.

  • 2. Microfluidic Device Fabrication:

    • o A microfluidic device with integrated microwires was fabricated using polydimethylsiloxane (PDMS). The device features a microchannel where water samples are introduced for testing.

    • o The microwires were installed perpendicularly to the microchannel to measure electrical resistance changes as bacteria bind to the CIP-coated surfaces.

  • 3. Bacteria Detection:

    • o Bacteria suspensions of known concentrations were flowed through the microchannel, and the electrical resistance between the microwires was measured before, during, and after exposure to bacteria.

    • o The resistance change is used to quantify the bacteria present, with specific attention to the normalized resistance changes (R1/R0 and R2/R0) to account for baseline variations.

2.3.2 Key findings

• 1. Detection Sensitivity and Range:

  • o The sensor demonstrated a dynamic range of 10⁴ to 10⁷ CFU/mL for E. coli detection, with a limit of detection (LOD) of 2.1 × 10⁵ CFU/mL and a limit of quantification (LOQ) of 7.3 × 10⁵ CFU/mL.

  • o The sensitivity of the sensor was calculated to be 7.35 µS per CFU/mL within the linear range.

• 2. Specificity:

  • o The sensor showed high specificity for E. coli, with significantly lower responses to non-target bacteria like Listeria and Sarcina, demonstrating the effectiveness of the CIP-based selective binding.

• 3. Comparison with Other Methods:

  • o The CIP-MW-based sensor offers advantages over traditional biological recognition methods (e.g., antibodies, aptamers) due to its stability and cost-effectiveness.

  • o The conductometric method used is simpler and more cost-effective compared to other electrochemical techniques, which often require complex instrumentation and reference electrodes.

• 4. Practical Applications:

  • o This sensor is particularly suited for field-deployable water quality monitoring, providing a real-time, durable, and affordable solution for pathogen detection.

  • o The study suggests potential improvements, including increasing the active surface area of electrodes and incorporating pre-enrichment processes to enhance sensitivity further.

The developed microfluidic sensor effectively captures and detects E. coli in water using CIP-coated microwires and conductometric measurements. Its high specificity, sensitivity, and low-cost fabrication make it a promising tool for on-site water quality monitoring. Future research will focus on addressing non-biological impurities, reusability of the sensor, and further optimization for field applications.

3 Integration of electrodes on the microfluidics-based biosensor

An electrode is a conductor that is used to connect electrically to nonmetallic components such liquids, gases, vacuums, and plasmas [96]. Electrode integration in microfluidic chips is used for a variety of applications, such as biosensing, electrochemistry, and cell stimulation [97]. In most cases, the electrode is used to carry current from one place to another. In a microfluidics device, an electrode is used to charge the particle inside the liquid sample to move within microchannels [98]. Silver, gold, platinum, and palladium, copper, nickel, and titanium are some of the unique metals utilized to create electrodes (see Table 2) [99]. Platinum is the costliest metal when compared to silver and gold, but it also makes the most stable electrode material since it degrades the least, can withstand high temperatures, and comes in a variety of sizes [100]. Platinum can be altered using aptamers and enzymes for biorecognition; this alteration aids in selective binding, which enhances performance. Silver is the least costly and most stable in air, although it tends to move over resistor surfaces in humid environments [101], the silver surface can be modified with DNA/proteins for better antigen detection. Gold is renowned for its great electrical conductivity and dependability, yet it is extremely difficult to solder and readily vaporizes into the substrate at a very low temperature, much like silicon [102]. Gold surfaces can be modified with enzymes, DNA, and antibody to improve sensitivity and specificity. Copper is an easily available and affordable material, known for high electrical conductivity. Copper surfaces modified with proteins and DNA have increased sensitivity and specificity for biosensing Although copper is an excellent material but has poor mechanical properties (e.g. hardness, elasticity, malleability, ductility, etc.) at higher temperatures [103]. Research discovered that to solve these problems, most copper electrodes are doped with graphene. Graphene is suitable for reinforcement in copper because of its high thermal conductivity, excellent mechanical strength, and electrical conductivity [104]. For better biorecognition, the graphene surface functionalized DNA and protein improves the conductive property, selectivity, and specificity. However, graphene suffers under intense electrochemical stress and tends to lose stability. Nickel metal is also used to make electrodes because is ductile, and resistant to corrosion, thermal stability, and hardness, they are great competitors to the platinum electrode because of corrosion resistance [105]. Despite the excellent advantages, nickel is expensive and tends to have low specific energy. For better use, Nickel surface can be functionalized with proteins such as histidine-tagged proteins to improve the specific binding. The last metal to discuss in this section is titanium, in most cases, titanium is used with platinum/palladium to make electrodes. Titanium is an excellent conductor of electricity and has improved efficiency for electrolytic reactions [106]. Titanium surfaces modified with TiO2NPs have increased performance sensitivity. The metals discussed are important for the creation of electrodes, the electrodes are used in electrochemical reactions where they are integrated into microfluidic chip channels, chambers, and valves where they help in converting the biochemical signal into electrical signals [107].

Table 2 Different types of materials used as electrodes for integration onto microfluidic biosensors with advantages and disadvantages

Electrodes are a crucial component of lab-on-chip systems and are employed in a wide variety of applications, including electrochemical sensing and electrokinetic transportation [108]. Over the last 30 years, different techniques for fabricating electrodes for microfluidic devices have been studied [109, 110]. A good method for making electrodes for microfluidic devices should be inexpensive, simple to make in a nonclean room setting, able to incorporate a range of high-quality materials and have enough resolution of a few tens of micrometers [111, 112]. This method includes injecting printing, Xurography, chemical etching, etc. (Fig. 9) [113, 114].

Fig. 9

Different methods for fabricating electrodes for microfluidic chip

To incorporate electrodes in microfluidic devices, a variety of techniques are used, such as photolithography, drop-casting, chemical vapor deposits, electrodeposits, and screen printing [115]. In the photolithography process, the electrodes are used to create patterns on the surface of the microfluidic chip, electrodes are typically deposited by depositing a layer of metal on the surface and etching away the unwanted portions of the layer [116]. In drop-casting, a dispersion of material is simply dropped onto the surface of the microfluidic device and dried [117]. In CVD, the material is synthesized directly on the surface of the microfluidic device [118]. In electrodeposition, the material is deposited onto the surface through an electrochemical process [119]. Screen printing is a method in which a thin film of electrode material is deposited onto the surface of the microfluidic device through a stencil [120]. The surface of the microfluidic device is covered with a stencil that has a pattern that matches the desired electrode shape. A mixture of electrode material and binder is then squeezed through the stencil and onto the surface [121]. The stencil is then removed, leaving a pattern of the electrode material on the surface of the microfluidic device [122]. Examples of applications of metal electrode-integrated microfluidic devices include biosensors for glucose or lactate detection, DNA sequencing, and microfluidic-based cell stimulation [123]. Scientific research, environmental monitoring, and medical diagnostics are just a few of the industries that have employed these devices [124]. The schematic design of the microfluidic device is shown in Fig. 10. The microfluidic device consists of two PDMS layers, with the first thin layer comprising the microfluidic channel and the gold electrode integrated into it [125].

Fig. 10

A three-dimensional illustration of the microfluidic module on a complementary metal–oxide–semiconductor chip demonstrates the metal-coated through-PDMS-vias [126]

4 Imprinting of Lab-On-Chip (LOC) platforms on microfluidic devices

Microfluidic lab-on-chip (LOC) platforms have received a lot of attention because they might potentially replace a conventional laboratory with all the necessary equipment [136, 137]. The present focus is on using small remote automated systems because typical analytical techniques are generally expensive and time-consuming, requiring many stages (such as sample, transport, and preparation) [138]. These LOC sensing systems have several well-known benefits, including compactness, minimal sample consumption, cheap manufacturing costs, improved overall process control, real-time analysis, and quick responses [139, 140]. These qualities make it possible to conduct real-time measurements on-site. The LOC also function in a way that allows sample pretreatment and chemical tests to be carried out there [141]. A microfluidic device is comprised of a set of fluidic unit operations, designed, and easily incorporated inside [142]. The use of a microfluidic device enables the parallelization, automation, and downsizing of (bio-)chemical processes in a generic and uniform manner [143]. The platform permits the rapid and adaptable execution of multiple tests, depending on the fabrication process. Microfluidic lab-on-chip platforms include centrifugal, capillary, electrokinetic, pressure-driven, and acoustic (Table 3) [144]. The platforms have different operation units which help in different applications see Fig. 11. Microfluidic units are used in capillary platforms to facilitate liquid flow; these units are generally essential components that complete the platform and enable optimal performance [145]. The platform’s main principle is the movement of a non-resistant liquid through the microstructure layer’s capillaries using capillary forces [146]. The liquid samples are poured into a micro-chamber and penetrate the underlying fleeces. Another approach is the use of a sample to directly fill the strip, which is commonly used for patients in self-testing [147]. For instance, in diagnostic testing, test strips are directly touched by the blood that has been spilt from a fingertip that has previously been punctured with a lancet. The complete blood sample is first filtered through a separate fleece within these test strips, which holds back the blood cells [148].

Table 3 Different types of microfluidic platforms with unit operations, advantages, and disadvantages for different applications

Fig. 11

A microfluidic chip designed for research on stem cells. Cell separation, detection and counting, migration or viability tests, and differentiation research all make use of several integrated microfluidic components and sensing modules [149]

4.1 Applications of microfluidic chip LOC platforms in biomedical analysis

Bioanalysis, which belongs to analytical chemistry, refers to the identification and quantitative measurement of biological components found in biological systems, including metabolites, proteins, bacteria, viruses, nucleic acids, cells, and other molecular biomarkers [156]. Many domains, such as disease diagnosis, cancer research, environmental monitoring, medication development, and food safety regulation, rely heavily on bioanalysis. Rapid identification of a variety of infectious diseases is made possible by the use of microfluidic chip LOCs in bioanalysis, which are used in point-of-care settings. The automation made possible by the microfluidic chip platforms eliminates the need for several steps in the diagnosing process [157]. The automation allows for sample preparations, analysis, and detection at a single device, which reduces long turnaround time. Researchers at UC Santa Cruz have created a unique chip-based antigen test that can detect SARS-CoV-2 and influenza A, the viruses that cause COVID-19 and the flu, respectively, with ultrasensitive during the COVID-19 outbreak [158]. This is a chip-based biosensor capable of detecting individual proteins one at a time, and we show how it can be used to detect and identify the antigens for multiple diseases simultaneously. Furthermore, in 2024 Researchers have also developed microfluidic platforms for oral, intraperitoneal, and inhalation-based delivery of insulin. Vacurebiotech researchers developed a microfluidic-based test kit HbA1c, which functions as a fluorescence immunoassay for the quantitative measurement of hemoglobin A1c (HbA1c) in human whole blood or peripheral blood [159]. It can help people with diabetes mellitus control and monitor their long-term glycemic condition. Moreover, food safety uses microfluidic LOC platforms to identify bacteria that cause diseases [160]. The major pathogens associated with food poisoning are bacteria, viruses, parasites, and fungi, these pathogen causes foodborne diseases. A testing approach utilizing microfluidic μPADs (paper analytic devices) was created in response to foodborne infections. The device is primarily utilized as an assay that provides a detection signal and data analysis, and it is equipped with microfluidic platforms for automation and sampling [161]. The bacteria responsible for foodborne illnesses are the focus of the chromogenic assay μPADs, which have been created and evaluated. Environmental monitoring is one area in which microfluidic LOCs are applied. Drug discovery is aided by microfluidic LOCs platforms. For example, Labcyte (later bought by Beckman Coulter) developed the Echo Liquid Handlers, a system that precisely transfers small amounts of liquids using microfluidic technology [162]. This platform is used in sample preparation, assay downsizing, and compound screening. The application of microfluidic LOC platforms is still in its infancy. The development of microfluidic organs on chip platforms, including as kidney, liver, lung, and intestinal chips, is still under investigation. In order to support livelihood, the systems seek to replace the composition and capabilities of human organs [163]. There are point-of-care diagnostic tools available for cancer biomarkers, infectious infections, and other ailments.

5 Materials used to fabricate microfluidic devices.

Microfluidic device design and fabrication for biosensors is challenging and frequently necessitates going over fundamental ideas [164]. The device’s wettability and biocompatibility are determined by the platform's dimensions, the materials selected, and the fabrication methods used [165]. In this section, the evolution of materials used for microchip fabrication is reviewed, including their advantages, disadvantages, and suitable applications are systematically discussed [166, 167]. As technology improves over time, various materials including polymers, composites, and paper have been used to create microfluidic biosensors (Fig. 12) [168]. In the last two decades, various materials have been introduced to fabricate microfluidic devices. There are some excellent reports on specific methods to fabricate microfluidic devices using specific materials [169, 170]. The melting point and heat conductivity of these materials should be high to withstand high-temperature reactions [171]. The microfluidic chip can be covered with thermal plaster or paste to further improve the material's thermal conductivity [172].

Fig. 12

Major materials for fabrication of microfluidic chip

In general, a wide range of materials, including silicone, glass, ceramic, paper, polymers, and thermoplastic can be used to create microfluidic devices (Table 4) [173,174,175]. Polymer materials continue to be the material of choice for microfluidic devices because they are inexpensive and simple to produce [176]. Polymers like polydimethylsiloxane (PDMS) and epoxy resin are popular materials because of their exceptional physicochemical and mechanical properties. Glass and silicon are also the most used building blocks for microfluidic devices such as chips and microreactors [177]. Table 4 highlights in brief the most prevalent materials and production processes utilized for the construction of microfluidic biosensor devices [178].

Table 4 Advantages and disadvantages of different materials for fabrication of microfluidic chip

5.1 Materials used to fabricate microfluidic chips

The fabrication of microfluidic devices utilizes a variety of materials, each selected based on the specific requirements of the application, such as chemical resistance, optical transparency, and mechanical properties. The choice of material significantly influences the device’s functionality, fabrication technique, and cost. Some of most used materials in the fabrication of microfluidic devices:

• 1. Silicon: Historically, silicon was the first material used in the development of microfluidic devices due to its well-established use in the semiconductor industry. Silicon offers excellent mechanical properties and thermal conductivity, making it ideal for applications requiring precise heat management and high durability. However, silicon is not transparent to visible light, limiting its use in optical applications, and it can be relatively expensive [26].

• 2. Glass: Like silicon, glass is frequently used due to its chemical inertness and optical transparency, allowing for easy observation of the fluids within the channels. Glass can be bonded to other materials like PDMS or another glass layer using methods such as anodic bonding. Glass is particularly favored in applications involving harsh chemicals or high temperature [179]. Integrating glass-based microfluidic chips on biosensors will produce high signal-to-noise ratios in the fluorescence method for biosensing. Using glass is challenging because it cannot withstand intense mechanical stress, it is fragile material to use and needs carefulness. It is also hard to fabricate fabricates using glass, the process involves complex multi-step process that requires trained personnel

• 3. Polymers: Polymers are perhaps the most widely used materials in recent microfluidic applications due to their versatility and cost-effectiveness are biocompatible materials, and they allow surface modification which makes integration of electrodes ease. The use of polymer to fabricate microfluidic chips improves the immobilization of biomolecules, this enhances the sensitivity and specificity of the biosensors. Common polymers include:

  • o PDMS (Polydimethylsiloxane): PDMS is the most popular polymer in microfluidics due to its flexibility, optical transparency, and gas permeability. It is particularly useful for biological applications as it is biocompatible. PDMS devices are typically fabricated using soft lithography techniques [180].

  • o Resin: Resin is a popular material choice in 3D printing, particularly used in processes known as Stereolithography (SLA) and Digital Light Processing (DLP). These methods leverage the properties of photo polymeric resin, which cures (hardens) in response to light exposure. It represents a powerful tool in the additive manufacturing arsenal, well-suited to applications that require intricate details and high aesthetic quality such as microfluidic devices [181].

• 4. Thermoplastics: Thermoplastics are biocompatible materials, are optically transparent, and have excellent mechanical properties such as flexibility, and durability. the use of thermoplastic to fabricate microfluidic chips for biosensing influences the sensitivity because they allow for surface medication, which includes modifying the surface with chemicals to it specific and sensitive, last is the integration of the electrode. Although thermoplastics are thermal stable, they tend to be affected at higher temperatures. Common thermoplastics include:

  • o PMMA (Polymethylmethacrylate), or acrylic, is another polymer used for its ease of machining and bonding, along with good optical clarity. It is often used in disposable device [182].

  • o COC (Cyclic Olefin Copolymer) and COP (Cyclic Olefin Polymer) are gaining popularity for their high chemical and thermal resistance, which is superior to many other polymers, and their low water absorption [183].

• 5. Paper: Paper-based microfluidics are a growing field, particularly for medical diagnostic applications in low-resource settings. Paper is inexpensive, disposable, and capable of wicking fluids through capillary action, eliminating the need for external pumps. It can be chemically modified to create hydrophobic and hydrophilic regions, guiding the flow of fluids [184].

• 6. Ceramics: Ceramic is also used to fabricate microfluidic chip for biosensing because of its mechanical strength, electrical insulation, chemical and thermal stability. The use of ceramic improves the sensitivity and makes the biosensor specific because is inert by nature and allows for surface modification and functionalization techniques that attach ligands, enzymes which target specific analytes. The mechanical strength makes it reliable to obtain reproducible and reliable results. although ceramic is mechanical strong, it cracks under intense mechanical stress

• 7. Metals: Metals are occasionally used in microfluidic devices for electrodes, heaters, or as a structural framework. Metals like platinum and gold are preferred for their conductivity and chemical stability [185, 186].

• 8. Hydrogels: Hydrogels are networks of polymer chains that can absorb large amounts of water. In microfluidics, they are used to fabricate valves and actuators due to their ability to change shape in response to changes in temperature, pH, or ionic strength. This makes them suitable for creating dynamic microfluidic system [187].

5.2 Materials challenges and considerations

Polymers such as PDMS are favored for their biocompatibility, optical transparency, and flexibility. However, PDMS’s hydrophobic surface can adsorb hydrophobic molecules, potentially leading to sample loss and contamination. PDMS also has issues with gas permeability, which can affect reactions involving sensitive or volatile compounds [188]. Materials such as silicon and glass offer excellent chemical and thermal stability, making them suitable for high-temperature processes and harsh chemical environments. Their main limitation is cost, and the complex fabrication processes required, which may not be feasible for disposable applications. Additionally, silicon is opaque to UV light, which can be a limitation for UV-based detection methods [189]. Other Polymers (other than PDMS): Thermoplastics like PMMA and COC are used for their ease of fabrication and lower cost compared to glass and silicon. Challenges include their lower solvent resistance compared to glass and silicon, which can limit their use with certain biochemical assays. Additionally, many polymers are not biocompatible, restricting their use in medical applications [179]. More details about the advantages and disadvantages of these materials can be found in Table 4.

5.3 Advantages and limitations of different microfluidic techniques

Techniques such as Capillary Electrophoresis are popular and well used. This technique allows for high-resolution separation and detection of biomolecules, making it highly effective for biosensing applications. Its advantages include low sample and reagent volumes, rapid analysis, and excellent separation efficiency. However, the technique is limited by the complexity of device fabrication and the need for precise control over operating conditions. Furthermore, CE devices can suffer from issues like sample adsorption and electroosmotic flow variability, which may affect reproducibility and sensitivity [190]. Another example technique is Droplet-based Microfluidics: This technique involves manipulating discrete micro-droplets independently, which can encapsulate single cells or molecular species for analysis. The main advantage is the high throughput and automation potential, reducing cross-contamination and reagent costs. Limitations include the technical complexity of droplet generation and manipulation systems, and challenges in droplet stability and merging [191]. Paper-based microfluidic devices use the capillary action of paper to transport fluids, offering a low-cost, portable, and disposable option suitable for field use and low-resource settings. While advantageous for their simplicity and accessibility, paper-based devices generally have lower accuracy and sensitivity compared to other microfluidic platforms, and they face challenges in multiplexing and quantitative analysis [192]. Some examples are shown in Table 3.

6 Limitations and challenges of microfluidic-based biosensors

While microfluidic biosensors offer significant advantages in terms of sensitivity, specificity, and the ability to handle small volumes of samples, they also face several notable challenges. These challenges include fabrication complexity, scalability issues, reliability, and integration challenges. Microfluidic biosensors often require intricate designs and precise manufacturing techniques, which can involve sophisticated and costly fabrication processes. The complexity of fabricating these devices with consistent quality is a major hurdle, particularly for mass production. Techniques such as soft lithography, while versatile, may not easily scale up and can suffer from issues like mold degradation and non-uniformity across batches [206]. In terms of scaling, the production of microfluidic devices from laboratory prototypes to commercial quantities remains a significant challenge. Many microfluidic devices are produced using methods suited for small-scale production, but these methods do not necessarily translate well to larger scales. Issues such as maintaining the precision of fluidic channels and the integrity of embedded sensors become exponentially difficult as production volume increases [26]. The reliability of microfluidic biosensors can be affected by several factors, including the variability in the fabrication process, the stability of biological components (such as enzymes and antibodies) within the devices, and the handling and storage conditions. Variability in channel dimensions, for instance, can lead to inconsistencies in fluid flow and sensor responses, impacting the accuracy and reproducibility of the biosensor's results [207]. Integrating microfluidic systems with electronic interfaces and data processing systems poses additional challenges. Effective communication between the microfluidic components and electronic readout systems is essential for the practical application of these biosensors. However, this integration is often complicated by issues related to size compatibility, signal interference, and the need for robust software that can process complex biological data [179].

There are ongoing efforts to address these challenges which include advanced manufacturing techniques such as developments in 3D printing and nano-fabrication technologies offer promising avenues to simplify the fabrication process and enhance scalability. These technologies can potentially lower the cost and improve the consistency of microfluidic devices [208]. Making material innovations is another approach to addressing these challenges. New materials that are easier to process and more durable are being explored. For example, the use of hybrid polymer-glass or polymer-metal systems might offer improved robustness and ease of fabrication while retaining the essential properties needed for sensitive biosensing [139]. Efforts to standardize microfluidic components and automate the assembly process are crucial for enhancing reliability and scalability. Standardization can also facilitate the integration of microfluidic devices with electronic components, reducing compatibility issues [209]. Robust Design and Quality Control: Implementing robust design principles and stringent quality control measures during the fabrication process can mitigate variability and enhance the reliability of microfluidic biosensors. This includes the use of precision engineering tools and real-time monitoring of production parameters [210].

7 Future directions and challenges of microfluidic technologies

The field of microfluidic biosensing is rapidly evolving, with ongoing research and technological innovations driving significant advancements. Discussing these emerging trends not only enriches the manuscript but also underscores the potential of microfluidics to revolutionize various aspects of healthcare, environmental monitoring, and biotechnology. One of the most significant trends is the integration of microfluidic biosensors with digital and mobile platforms. This integration facilitates real-time data analysis, remote monitoring, and enhanced connectivity, making it possible to deploy these devices in telemedicine and personalized healthcare applications. Researchers are exploring ways to connect microfluidic devices with smartphones, creating portable, user-friendly systems that can perform complex analyses with minimal user input [211, 212] The development of wearable microfluidic biosensors is another exciting trend. These devices are designed to be worn on the body and continuously monitor physiological parameters or detect specific biomarkers in sweat, tears, or interstitial fluids. This ongoing research aims to make health monitoring more proactive and predictive, with applications ranging from fitness tracking to early disease detection [213,214,215]. Future microfluidic biosensors are likely to feature enhanced multiplexing capabilities, allowing them to detect multiple analytes simultaneously. This is particularly important in complex sample matrices, where the ability to assay several targets at once can significantly improve diagnostic accuracy and speed. Additionally, integrating various sensing modalities (electrochemical, optical, and acoustic) within a single device could provide comprehensive analysis tools that are both compact and versatile [216]. Advancements in materials science are crucial for the next generation of microfluidic biosensors. Researchers are experimenting with novel materials such as graphene, conducting polymers, and nanocomposites to enhance the sensitivity and functionality of biosensors. Furthermore, the adoption of innovative fabrication techniques like 3D printing offers the potential for rapid prototyping and customization of devices at a lower cost and with greater design flexibility [217, 218]. The integration of artificial intelligence (AI) and machine learning (ML) algorithms with microfluidic biosensing technologies is a growing area of interest. AI and ML can significantly enhance data processing capabilities, improve the accuracy of diagnostics, and enable predictive analytics. This trend is particularly promising in the context of complex biological systems where dynamic responses are required for decision-making processes [219,220,221]. Microfluidic biosensors are increasingly being applied beyond the medical and healthcare sectors, extending into environmental monitoring and food safety. These devices can detect pollutants, pathogens, and toxins with high sensitivity and specificity, offering a portable solution for on-site testing. This application area is expected to grow, driven by global demands for safer environments and food supply chains [222,223,224,225,226]. The integration of cell-imprinted polymers with microfluidic sensors also opens new avenues for biosensing technologies. These systems offer significant advantages in terms of specificity, sensitivity, and miniaturization, making them ideal for on-site testing and real-time analysis. Future research should explore the scalability of these technologies and their integration into global health diagnostics and environmental monitoring systems. Integrating discussions on these cutting-edge research directions not only fills a significant gap in the literature review but also enriches the overall discourse by shedding light on innovative approaches that could lead to transformative changes in biosensing technology [93, 95].

8 Conclusion

In this work, we review the current progress in microfluidic applications, specifically focusing on biosensors for disease detection. The work dives into the use of microfluidic chips for biosensing, integrating electrodes on the microfluidic chip for biosensing, microfluidic platforms that enable microfluidic devices to allow separation, mixing of liquids, recent advances in microfluidic device on technology, focusing on bioanalysis and materials used to fabricate microfluidics chips and the importance of those materials. The use of microfluidic chips for biosensing improves the performance of the biosensors and adds the advantages of automation, small sample use, and high throughput. Another compartment that improves the performance of microfluidic devices is an electrode, electrodes used to charge the particle inside the liquid sample to move within microchannels. The integration of the electrodes in the microfluidic device impacts the flow dynamics of the samples positively by increasing flow speed. Microfluidic devices are fabricated using different materials which plays a crucial role in the functioning of the device. Recently, microfluidic chips have been used in many fields of science, including chemistry, physics, biology, and engineering. The use of microfluidic devices encouraged more research and technological improvements. There is possibility of microfluidic device expansion due to the current growth and attention given to microfluidic devices on biosensing. Microfluidics offers a great promise in the development of highly sensitivity diagnostic technologies. As technology develops the integration of microfluidics and other technologies such as machine learning can help in the development of POC devices which can be useful in resource limited countries.