A Progress Review of Multiplex Biosensors with Microfluidic Chip

Abstract: Utilizing multiplex biosensor could greatly increase test throughput and the amount of information obtained in a single assay. As one of micro-total analysis system (uTAS), microfluidic chip has been widely used in biosensors for high-throughput analysis. In this review, the recent progress of all kinds of biosenors were reviewed, including optical, electrochemical, and some others, which integrated with microfluidic chip for simultaneous detection of multiple analytes.

Keywords: Microfluidic chip; Sensor; Multiple detection.

1. Introduction

The simultaneous sensor of multiple analytes is appealing for many applications. Multiplex biosensors have many advantages over single-analyte assays, which increase the test throughput and improve test efficiency. There has great enthusiasm for developing multianalyte analysis using parallel single-analyte and simultaneous multianalyte based on optical detection methods ^[1] and electrochemical immunosensors (EIS) detection strategies ^[2]. Among standard technologies, (bio)sensor technology allows to design ad hoc analytical systems which include a combination of advantages, in terms of selectivity, reduced time-to-result, miniaturization, and consequently portable system platforms for a single analyte as well as simultaneous multi-analyte testing. Technological advancements in the fields of microelectromechanical systems (MEMS)[ref], material science[ref], rational design[ref], microfluidics[ref], and sensor printing have radically shaped biosensor technology.

Microfluidic chip technology is a technique for manipulating microfluidic fluids in a micrometer channel [ref]. There have been considerable efforts in recent years to couple biosensing with microfluidics, benefitting from the small sample volumes, easy to multiplex and integrate, rapid turnaround times and high portability offered by microfluidics ^[4]. Therefore, microfluidic chips is one of the most striking technology with which more and more biosensor systems have been integrated to improve the overall performance of the sensing system ^[3]. In this review, the basic concepts and recent development in the multiplex biosensors with microfluidic chip were presented. This review was divided according to the different kinds of transducing element including electrochemical, optical, magnetic and acoustic sensors which have been used into microfluidic based multisensors. For this review article, we have collected examples mainly from the literature from 2010 onwards that involve microfluidic multiplex sensors for biological application. Due to the quick development of manifold technology and cross infiltration of different technology, it is difficult to avoid the unsuitable classification and missing some papers. The scientific literatures are replete with in-depth reviewing of one specific topic related with microfluidic based multisensors[6-16].

2. Multiplex Electro-sensor with Microfluidics

Being one typical sensor, an electrochemical sensor measures the (bio)chemical reaction through a digital electronic signal converted by the transducer. As both an immobilization and transduction platform, the electrode have been used by most of electrochemical detection. High chemically or biologically sensitive  e layers which can recognize the target analyte through immobilization of antibodies,enzymes,or a short oligonucleotide sequence at the electrode ^[18,19]. . For example, a redox molecule produced by the combination of target molecular with an enzyme-labeled secondary biorecognition probe, can diffuse to the electrode surface, and thenresulting in a measurable signal ^[20]. Electrochemical detection methods reduce maintenance costs with high selectivity and sensitivity, allowing low levels of targets detection without prior sample treatments. At the same time, in comparison with optical methods, electrochemical techniques could be easily integrated into miniaturized systems, which fulfil the requirements of microfluidic technology and their POCT (Point-of-care Testing) application. Achieved through microfabrication technology, multiple electrochemical sensor integrated with microfluidic chip has attracted considerable attentions, as an  outstanding candidate for miniaturization [21–24].

2.1.1 Microfluidic chip integrated multiple electrochemical sensor with multiple labels

Multilabel assays ^[25]and spatially resolved assay systems with single label ^[26, 27] are two main traditional strategies to realize multi-target sensoring in electrochemical biosensors integrated with microfluidic .

Multianalytes recognized simultaneously by tracers with multiple labels have been proposed in electrochemical immunoassay. Simultaneous multianalyte electrochemical immunoassay was achieved using thionine-labeled and ferrocene-labeled antibodies for a-fetoprotein (AFP) and carcinoembryonc antigen (CEA) detection.  Each immune-recognition event yielded a distinct voltammetric peak with position and strength reflecting the identification and level of the corresponding antigen[29]. Simultaneous electrochemical immunoassay of AFP and human epidermal growth factor receptor type-2 (HER-2) was reported using metal-containing nanomaterials confined in the ordered mesoporous carbon matrix (OMC-M) as labels [30]. However, it is not easy to find proper multilabels and  the throughput is always limited by the number of labels available. As a result, single-label methods for detecting multiple analytes can provide simplified alternative to multilabel methods.

2.1.2 Microfluidic chip integrated multiple electrochemical sensor with single labels

2.1.2.1 Electrochemical sensor array in the same unit

Single-label methods for multiple analytes can offer advantage of simplicity. Butdiscrimination between different detection events need to be carefully considered in single-label methods. One approach to help discriminate is using an array of electrodes allowing simultaneous recognition of multiple targets at the local regions immobilized with the corresponding antibody or oligonucleotide [ref]. The method of sensor array also has been developed as the main mode to perform multiple electrochemical sensor in microfluidic chip.

For example, it was shown that by carefully controlling the electrode surface pre-treatment and derivatisation with thiolated antibodies or short DNA probes on different electrode arrays, the detection of several key health parameters on a single chip was achievable ^[28]. Simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in microfluidic electrochemical sensor was achieved with another microfluidic electrochemical immunoassay system. Hypersensitivity at sub-pg mL^-1 was achieved by using off-line capture of analytes and capture antibodies attached to an 8-electrode measuring chip^[31]. It was also reported that electrode modified with the diazonium functionality allowed selectively immobilizing different biomolecules and then facilitating multianalyte detection ^[20]. A low-cost reusable microfluidic electrochemical sensors platform integrated ion-selective microelectrodes (μISE) for dynamic online monitoring of cell culture parameters , such as dissolved oxygen (DO), and hydrogen (H⁺), sodium (Na⁺), and potassium (K⁺) cations in the input and/or output of a cell culture chamberwas developed [32]. With the use of an internal marker and then no need of washing steps, a multiplex electrochemical sensor with improved sensitivity and simple operational procedure was given. A hair pin probe with a porphyrin tag was immobilized on an array of gold sensors for the simultaneous detection of three bladder cancer biomarkers with a detection limit of 250 fM in 20 min^[94].

Figure 6. Design of the microfluidic sensor. (A) Image of device used with an array of 20 sensors including a magnified image of an individual sensor with working electrode, reference electrode and counter electrode, scale bars indicate the length scale. (B) Schematic of the electrode device and the microfluidic channel design including 6 parts [94].

It has been demonstrated that multi-analyte analysis is possible in single labeled system, provided that the sensing electrodes have sufficient separation to prevent signal interference (cross-talk) between neighboring electrodes [20,28,32]. However, it is noteworthy that when substances have to be detected simultaneously, it is often unavoidable that an electrochemical sensor array in the same unit suffers from crosstalk potentially occurring due to the diffusion of electroactive product between neighboring electrode [33, 34].

In order to reduce the interference from cross-talk, the spatial separation of the electrodes technique was employed to enable individual immunoassays to be performed separately [35]. A microarray consisting of three closely spaced Au disk electrodes in 500 mm diameter was selectively functionalized with antibody probes for thecytokines TNF-a, IL-12, and IL-1b detection [36, 20]. A microfluidic device consisting of two separated gold electrodes deposited on a porous nylon membrane was reported for qualitative detection of antiferritin and streptavidin. [37]. However, above method is only workable in the case where free electrochemical products are generated only in close proximity to the electrode surface or the lateral movement and contamination of adjacent electrodes are minimized. The background signal inherent in this assay design will increase as the number of analytes increases [38].

In an array of immunosensing electrodes, as an excellent material enabling the highly efficient capture of enzyme-generated product by the electrode, IrOx matrix was used leads to less cross-talk between sensors and facilitates the development of miniature devices for the measurement of four analytes, GIgG, MIgG, HIgG, and CIgY[39]. The immobilization of the electrontransfer mediator on individual immunosensors to shuttle electrons ^[40,41] or the incorporation of reagentless electrochemical immunosensors ^[42,43]were also adopted to completely avoid the electrochemical crosstalk during simultaneous multianalyte testing. A reagentless immunosensor array was fabricated by embedding 4 kinds of HRP-Ab-Au nanoparticles in a newly designed biopolymer/sol-gel matrix formed on screen-printed carbon electrodes (SPCEs). The electrical communitcation became easier due to the cooperation effort of sol-gel matrix and gold nanoparticles. The presence of nanoparticles also improved the permeability for the analyte to penetrate into the sol-gel film for immunoreaction. Due to increasing spatial blocking, the electrochemical responses decreased, leading to a reagentless immunosensing without cross-talk ^[44,41]. Membrane was introduced into chip to limit the diffusion during multiple electrochemical analyses. The performance of an integrated system with thin-film electrode arrays was proved with enhanced the efficacy and detection limit [45]. Recently, nanoporous membrane based electrochemical sensor has been employed due to its enhanced sensitivity and easy fabrication process. A non-biofouling polyethylene glycol (PEG) based microfluidic chip integrated with functionalized nanoporous alumina membrane was reported for simultaneous electrochemical sensoring of two types of bacteria Escherichia coli O157:H7 and Staphylococcus aureus in mixture [46].

There are also reports that the multiple assays were executed in a sequential manner to avoid cross-talk [47, 48]. The reproducible detection of D-glucose and D-fructose detection was executed consecutively in the same microreactor by a specific sequence of potentials applied at the reservoirs  each assay [47]. In a microfluidic chip-based online electrochemical system (OECS) to realize in vivo continuous and simultaneous monitoring of glucose, lactate, and ascorbate in rat brain, the SWNT-modified electrode in the upstream channel and paralleled glucose and lactate biosensors in the downstream channels were carefully aligned ^[48].

2.1.2.2 Electrochemical sensor array in independent units

However, cross-talk between nearby microelectrodes can still become problematicespecially when the biosensors’ principles are similar. To complete avoid of cross-talking during multiassay which is the main problem in the electrochemical array [49-51],  address-dependent sensing platform, for example, parallel single-analyte assays channels [21, 38,54, 55,56,60,61] or independent chamber were used[24,59.

There is report that detailed the fabrication and use of a plastic multilayer and three-channel microfluidic fixture. Nine individually addressable sensing elements for multianalyte DNA detection and an integrated resistive heater were fabricated together ^[55]. An integrated different nanoband electrode array, such as copper, platinum, gold, etc., in different channel with diverse buffer solution respectively was utilized for amperometric detection of glucose and metabolites in serums from diabetics, including aldehyde compounds (glyoxal and methylglyoxal) and short organic acids (lactate, urate and 2-hydroxybutyric) ^[56]. Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules was reported. These electrodes were premodified by different kinds of aptamers to selectively capture the corresponding target ^[61]. A cyclo-olefin polymer (COP) microfluidic chip that integrated with six independent measuring channels allowed the simultaneous or sequential performance of multianalyte measurements on the same chip ^[21]. Three influenza viruses, H1N1, H5N1, and H7N9, were successfully detected from three separate sensing regions ^[38].

A cheap alternative to fabricate individually addressable gold electrode arrays through microwells fabricated on commercial gold compact discs-recordable (CD-R) was described. After the integration into a simple microfluidic device, this system was used to detect a cancer biomarker protein [60]. A multi-channel poly (methyl methacrylate) (PMMA) microfluidic biosensor with interdigitated ultramicroelectrode arrays (IDUAs) for was reported. As proof of concept, five microchannels were constructed in parallel on a large single IDUA for simultaneous amperometric detection of potassium ferri and ferro hexacyanide with comparable reproducibility to those obtained with separate single channel devices. The ability to be able to chip was quantify the specific nucleic acid sequences using a sandwich approach within 250 s and with a limit of detection of 12.5 μM was also demonstrated subsequently^[54].

Recently, a simultaneous microfluidic electrochemical biosensor composed of five chambers connecting to each electrochemical sensor to detect multiple biomarkers of pulmonary hypertension diseases in a single device was demonstrated^[24]. Scanning electrochemical microscopy (SECM) has been used as an amperometric biological imaging tool[57,58].Based on complementary metal-oxide-semiconductors, addressable devices with micro/nanoelectrode arrays on chips have been developed to obtain images in different areas with higher temporal resolution. This integrated amperometric sensor (Bio-LSI) was used for the simultaneous and continuous detection of human immunoglobulin G (hIgG) and mouse IgG (mIgG). Hydrogen peroxide enzymatically produced by the glucose oxidase captured at intersections was detected simultaneously by 400 microelectrodes of a Bio-LSI chip[59].

Figure 2. a) Photograph of the microfluidic channel network and its different sections. b) Cross-sectional view of one channel [53]. c). Schematics of fixture structure. Black in the cross-section views indicates fluid. [55].

2.2 Electrogenerated chemiluminescence

In electrogenerated chemiluminescence (ECL), the label emits detectable light in response to electrical current. Without optical excitation which avoids background interferences from scattered source light or sample autofluorescence, the detection limits of ECL are extremely low. Due to the limited number of available labels in a single run, multianalyte (array) ECL assay with one universal label to detect all analytes on different spatial areas, is regarded as more suitable for high-throughput multianalyte than multi-label mode [12]. A hydrophobic polymer microwell ECL array incorporated with antibody-coated single wall CNT (SWCNT)  was reported for multiple protein detection. The array base was a simple pyrolytic graphite block connected to a potentiostat, without the multielectronic fabrication. LOD for interleukin-6 and PiSA on the ECL arrays were equivalent to or better than commercial bead-based protein measurement systems. This array is readily adaptable to interface with microfluidics for simultaneous detection of up to 10 proteins [62]. A disposable ECL immunosensor array was constructed by multiple antigens on different WEs of the screen-printed carbon electrode (SPCE) (SPC) substrate. In a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to bind with their antibodies labeled with tri (2,2′-bipyridyl) ruthenium(II) [64]. The ECL signals in the array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. And then, as results of the ECL readout mechanism and the sequential detection mode, cross-talk was avoided between adjacent immunosensors [64-66]. One kind of rapid multiple assay of cancer cell surface biomarkers was reported by a chip microarray platform using electrochemiluminescence resonance energy transfer (ECL-RET) strategy. The platform was characterized by consisting of 64 antigen-decorated CdS nanorod spots with the diameter of 1.0 cm uniformly distributed on 16 ITO strips [63].

2.3 Electrochemical impedance spectroscopy (EIS)

Electrochemical impedance spectroscopy (EIS) is a rapidly developing electrochemical technique for the characterization of biofunctionalized electrodes and biochemical events at electrode surface. EIS can provide information of various biochemical events occurring in biosensors, which directly or indirectly alters the electrical properties of the sensor surface^[91][92]. At the same time, impedance spectroscopy has the potential for label-free integrated electrochemical detection in microfluidic lab-on-a-chip applications. A microfluidic chip for multiplexed detection of bacterial cells that used antimicrobial peptides (AMPs) as specific biorecognition elements for bacterial cell was presented. The AMPs were immobilized onto an electrical impedance microsensor array. Samples containing Streptococcus mutans and Pseudomonas aeruginosa were detected at minimum concentrations of 10⁵ cfu/mL within 25 min ^[93]. A microfluidic impedance device capable of – both the flow ratio sensing and the conductivity difference detection between the stream of sample and reference buffer was presented. With a flow focusing structure, in which the core flow has a higher conductivity than the sheath flow, the conductance of the device varied linearly with the flow ratio. If deionized (DI)-water sheath flow was used as a reference, the difference in conductivity between the  core buffer flow and sheath DI-water could be detected with a detection sensitivity of up to 165 nM of sodium chloride solution^[97].

2.4 The summary of multiple electrochemical sensor integrated with microfluidic chip

Recent advances in multiple electrochemical sensors integrated with microfluidic chip divided according to different kinds of transducer were summarized in Table 1.

Table 1. Recent advances in multiple electrochemical sensors integrated with microfluidic chip divided according to different kinds of transducer.

Methods and Analyte	LOD and/or Linear range	Ref.
Amperometry
Protein and DNA	<1 ppt	[20]
Goat IgG, mouse IgG, human IgG, and chicken IgY measurement	3 ng/mL for all analytes	[35]
Up to four different analytes	40 mM glucose	[55]
DNA sequence	12.5 μM	[54]
Glucose and lactate	A detection limit of glucose and lactate down to 10−7  and 10−6 M	[78]
Hb and HbA1c	The dynamic ranges for Hb and HbA1c were 1 × 10−4 to 1 mg mL−1 and 1.83 × 10−3 to 18.3 mg mL−1	[75]
Human immunoglobulin G (hIgG) and mouse IgG (mIgG)	In the range of 0.01–1.0 µg mL-1	[59]
Glucose and creatinine in blood serum	Glucose and creatinine can be measured at 2–8mM and 10-2 to 10 mM	[89]
S. enterica subsp. enterica serovars Typhimurium and Choleraesuis	The discrimination of S. Typhimurium from S. Choleraesuis in blood containing between 8 × 102 and 6.9 × 104 CFU/mL	[107]
Differential pulse voltammograms
CA 19-9 and CA 125	0.2 U/ml for CA19-9 and 0.4 U/ml for CA125. The linear range of CA19-9 is 0–24 U/ml with a correlation coefficient of 0.9992 and CA125 is 0 to 25 U/ml with a correlation coefficient of 0.9953	[41]
Cancer markers ( ACTB, PLAUR, EGFR, ERBB2 and ESR1)	The limits of detection approximated 3 nM of the corresponding sequence. Good linearity exhibited in the concentration range 3–20 nM.	[28]
Carcinoma antigen 153, carcinoma antigen 125, carbohydrate antigen 199, and carcinoembryonic antigen	Carcinoma antigen 153 : 0.2 kU/L, carcinoma antigen 125: 0.5 kU/L, carbohydrate antigen 199: 0.3 kU/L, and carcinoembryonic antigen:0.1 g/L	[44]
Chronoamperometry
Simultaneous detection of IL-6 and PSA	Detection limits for PSA and IL-6 were 0.23 pg mL−1 and 0.30 pg mL−1 .	[31]
Glucose and lactate	A sensitivity of 157 ± 28 nA/mM for the glucose sensor and 79 ± 12 nA/mM for the lactate sensor was obtained.	[51]
H1N1, H5N1, and H7N9 virus	The detection limit was as low as 1 pg/ml of each virus. The linear range was 1–10 ng/ml.	[38]
Tumor Markers (carcinoembryonic antigen, α-fetoprotein, β-human choriogonadotropin, and carcinoma antigen 125)	Simultaneous detection of three targets in clinical serum samples with concentrations up to 188 g/L, 250 g/L, 266 IU/L, and 334 kIU/L, respectively. The detection limits were 1.1 g/L, 1.7 g/L, 1.2 IU/L, and 1.7 kIU/L.	[40]
Glucose, Lactate, and Ascorbate	The basal levels of glucose, lactate, and ascorbate were 0.42 ± 0.05 mM, 0.97 ± 0.07 mM, and 11.2 ± 1.5 μM (n = 3), respectively.	[48]
Seven tumor markers: AFP, ferritin, CEA, hCG-β, CA 15-3, CA 125, and CA 19-9	The detection limit was <2 ng/mL (or units/mL) for most analytes	[33]
ATP and cocaine as the two model of small molecules	The detection limit was 3 × 10-10 M for ATP, 7 × 10-8 M for cocaine.	[61]
Hydrogen, potassium and calcium ions	There was a near-Nernstian responses with slopes of 62.62 mV±2.5 mV pH-1, 53.76 mV±3 mV-log[K+] -1 and 25.77 mV±2 mV-log[Ca2+] -1 at 25 ℃±5 ℃, and a linear response within the pH range of 2–10, with potassium and calcium concentrations between 0.1 M and 10-6 M.	[71]
Multianalyte DNA detection		[55]
Troponin T	Detection limit of 0.017 ng/mL in PBS. while the clinically relevant range is 0.05–1.0 ng/mL.	[21]
Cyclic voltammetry
Interleukin-6 (IL-6)	The detection limit of IL-6 in diluted serum was low at 10 fg mL−1 (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL−1	[60]
HRP and bisphenol A	The detection limitsfor horse radish peroxidase (HRP) and bisphenol A assays were 12.5 ng/ml (2.84×10-10 M ) and 10 ng/ml (44×10-9 M).	[45]
Glucose, dopamine, and uric acid in human serum	An LOD of 0.05 × 10−3 m for glucose with the linear range in0.05 × 10−3 to 6 × 10−3 M with . An LOD of 0.5 × 10−6 M for DA with the linear range in 1 × 10−6 to 200 × 10−6 m. An LOD of 5 × 10−6 m for UA  with the linear range in 5 × 10−6 to 400 × 10−6 M.	[79]
Urea and creatinine	Linear ranges (R2=0.9875 and 0.9907) in the concentrations from 3.16 10-4 to 3.16 10-2 M and 3.16 10-3 to 3.90 10-2 M can be obtained in the urea and creatinine.	[104]
Dissolved Oxygen (DO) and Hydrogen (H+), Sodium (Na+) and Potassium (K+) cations	A linear range of 0 and 8 mg/L for oxygen. A linear response in range between pH 9 and 3 with a slope of 60.7 mV/pH for H+ μISE. A linear response to the activity of Na+ over the range in 1 x 10-5 M and 1 x 10-1 M with a slope of 55.19 mV/decade, and the detection limit presented was 5 x 10-7 M. For the K+ μISE, a linear range between 3 x 10-5 M and 1 x 10-1 M with a slope of 50.98 mV/decade.	[32]
Impedance spectroscopy
Mixed bacteria of E. coli O157:H7 and S. aureus	A linear detection range from 102 CFU/mL to 105 CFU/mL with the limit of detection (LOD) around 102 CFU/mL	[76]
Two types of cancer cells (CCRF-CEM and Ramos cells)		[77]
Two common human pathogens, Streptococcus mutans and Pseudomonas aeruginosa	Both pathogens were detected at minimum concentrations of 105 cfu/mL	[93]
NT-proBNP and cTnI	The detection limits of NT-proBNP and cTnI were 0.33 pg/ml and 1.43 pg/ml	[199]
Square-wave voltammetry
Cardiac troponin I (cTnI) and C-reactive protein (CRP)	The linear range of this assay was between 0.01 and 50 g/L and 0.5 and 200 g/L, corresponding to cTnI and CRP.	[76]
Cyclic voltammetry and square-wave voltammetry
Four pulmonary hypertension-associated biomarkers, fbrinogen, adiponectin, low-density lipoprotein, and 8-isoprostane	They can be measured at 1 µg/mL concentration	[24]
Electrophoresis and Amperometry
Glucose, aldehyde compounds and short organic acids	These targets responded linearly in the concentration range of 10–2000, 1–500 and 5–600 mM, with the LODs of 4, 0.5 and 3 mM.	[56]
Electrochemiluminescence
Cancer cell surface biomarkers including AFP, CEA and PSA.	The linear ranges were 0.75 ng/mL to 75 ng/mL for AFP, 0.57 ng/mL to 0.50 μg/mL for CEA, and 1.0 ng/mL to 1.25 μg/mL for PSA.	[63]
Human, rabbit and mouse immunoglobulin Gs	The linear ranges for them were 10–400, 20–400, and 20–400 ng/mL. The detection limits were 2.9, 6.1 and 6.5 ng/mL (S/N = 3)	[64]
Human IgG and rat IgG	The detection limits were estimated to be 8.9 and 7.2 ng mL-1 for HIgG and RIgG (S/N=3). The linear range of concentration was from 20 to 400 ng mL-1.	[65]
PSA and IL-6	The detection limit (DL) for PSA was 1 pg mL-1 and for IL-6 was 0.25 pg mL-1(IL-6) in serum	[62]
miRNA-143	A limit of detection is 1.5 femtomoles	[173]

2.4 Nano-bioelectronics based multiplex sensors with chip

Nano-bioelectronics represent a rapidly expanding interdisciplinary field that combines nanotechnologies featuring nanostructures and nanomaterials with bioelectronics. They offer the potential to overcome existing challenges in bioelectronics, by not only helping reducing electronic transducer dimensions to the nanoscale, but also bringing significant improvements in the sensitivity ^[17]. For example, since redox-cycling electrochemistry critically depends on the miniaturizatioin and integration of closely spaced electrode systems, suitable nanoscale devices are very important for the development of sensor technology. A circular shaped nanocavity sensor arrays with a faster response time due to minimized maximal distance between access hole and sensor volum was developed, Redox-cycling concepts using nanocavity sensor arrays was also highlighted as providing an efficient amplification strategy for spatiotemporal detection of redox-active molecules [39].

2.4.1 Nanopore sensors

Nanopores ,a well-established class of label-free sensors, are capable of detecting single molecules by measuring the current disruption caused by the nanoscale aperture translocation of biomolecules such as DNA and proteins that are electrophoretically driven through the pore [67,68].As the youngest member of single-molecule detection techniques, nanopore sensor is low cost, typically requires low sample volumes and is easily scalable for high-throughput assay [69].  Keyser group reported the first example of multichannel ionic current detection of single molecules with 12 out of 16 glass nanopores embedded in PDMS grooves [70]. Compared with instable biological nanopore, solid-state nanopores have attracted increasing interest along with the development of micro- and nanofabrication techniques. These arrays can be produced in mass using advanced semiconductor processes which can further improve the cost and scale of analysis [71]. In order to improve the detection of nanopore sensors, an integrated patch-clamp system with sensitivity was proposed for for high-throughput single-stranded DNA analysis. There are two main blocks in this CMOS fabricated system with hardware simplicity. One is for amplification composing of three stages. Another compensation block was set up in order to minimize the deleterious effects brought by the input-offset voltage and the input parasitic capacitances ^[71]. The resistive-feedback trans-impedance amplifier (rf-TIA) for ionic current amplification, a commonly used element in nanopore sensor, occupies large areas and limits the number of sensors that can be integrated. A novel pseudo-resistor technique with a deep N-well NMOS transistor to reduce the feedback resistor size was fabricated in a silicon chip. Almost 90% reduced active area and an increase near 10-fold in the number of channels was attained ^[73]. Another kind of controlled dielectric breakdown (CBD) in solid-state membranes have been successfully used in scalable production of independently addressable nanopore arrays integrated within PDMS microfluidic devices. By confining the electric field within the microfluidic architecture, electrical noise in localized nanopore fabrication is significantly reduced. Multiplexed analysis of DNA or protein samples could be realized in different microfluidic channels on a single device [68].

Figure 3. Cross-section schematics of (a) a five-channel device and (c) achannel in a device with a micro-via layer confining the electric field and electrolyte to a precise location on the membrane (images not to scale). A second electrode (dashed line in (c)) can be added to produce a symmetrical electric field in the independent (top) channel. (b) and (d) Reflected optical images under a stereomicroscope of devices with five microfluidic channels situated directly on a SiNx membrane and isolated from the membrane by a micro-via layer, respectively. The white dashed lines in (b) and (d) indicate the orientation of the crosssectional views in (a) and (c), respectively.

2.4.2 Nanotubes and nanowires based sensors

Thanks to the exceptional properties of charge transport, electrical controllability, size compatibility with biomolecules and chemical-friendly surface [17,74], nanotubes and nanowires have been incorporated in sensing platforms. Dual CMOS polysilicon nanowire sensors were integrated with a microfluidic device to carry out on-chip based whole blood processing and simultaneous detection of multiple analytes. Reliable performance is demonstrated using an alpha-hemolysin protein nanopore 1.5 nm-diameter for detection of individual molecules of single-stranded DNA. The nanowire sensors permit the label-free and dynamic detection of multiple analytes. The microfluidic chip system will be further refined with single or multiple nanopores for advanced DNA analysis[75]. There are reviews concentrating on silicon nanowire sensors [17] and microfluidic-integrated nanotubes-/nanowires based transistors ^[74].

2.4.3 Nanomaterials combined electroanalytical sensors

Combining unique electrocatalytic properties of nanomaterials with electroanalytical techniques has become a rising field for sensing ^[44, 40]. For example CdTe and ZnSe quantum dots were conjugated with antibodies for sandwich immunoassay. After the CdTe and ZnSe were, After that, the square-wave anodic stripping voltammetry detection of Cd²⁺ and Zn²⁺ from dissolved quantum dots enabled the quantification of the two biomarkers of cardiac troponin I (cTnI) and C-reactive protein ^[76]. In a microfluidic chip integrated electrochemical system for in vivo continuous and simultaneous monitoring of glucose, lactate, and ascorbate in rat brain, single-walled carbon nanotubes (SWNTs) was used to facilitate the electrochemical oxidation of ascorbate. SWNTs  were also used as electrocatalyst during the oxidation of dihydronicotiamide adenine dinucleotide (NADH) by ^[48].. Au nanoparticles (AuNPs) with high electron-transfer capability were also coupled with target cells through aptamers to reduce the resistance of the system ^[77]. The platinum nanoparticles (Pt NPs) modified organic electrochemical transistors sensors demonstrated high sensitivity with a detection limit of glucose and lactate down to 10^-7 and 10^-6 M, respectively. The integration of this sensor with PDMS microfluidic channel provided a compact size, a short detection time and a low consumption of analyte ^[78].

2.5 New electro-sensor technology integrated with chip

The quick development in the field of electro-sensor, including printing technology, field-effect transistors and digital chip etc., also bring many new opportunities in microfluidic chip based multicomplex sensors.

2.5.1 Printing electrochemical sensors

How to reliably integrate nanoscale devices into chip at large quantities is one of the challenges facing for developing advanced sensor technology.New types of electrochemical chip comprising planar electrodes fabricated by thin film electrodes, self-assembled nanoparticles or screen printed electrodes (SPEs) have now gained widespread attention and rapidly replace conventional fabrication techniques. Self-assembled nanoparticles into porous layers, as the initial step toward bottom-up fabrication of nanoscale devices, still relies on costly thin-film deposition techniques. In this respect, multilayer printed electronics by special additive such as inkjet manufacturing can be an interesting alternative to classical micro- or nanofabrication including soft lithography, spincoating, and sputtering. The main advantage of this technology is the rapid prototyping capability. It can operate in a roll-to-roll mode. In addition, cost are reduced by site-selective deposition at desired location without the lift-off process ^[39]. In order to create a sensor chip not only easy, rapid and cheaper to fabricate but also has a smaller imprint area with good electrochemical sensing properties, an array consisting of eight working electrodes with shared Au quasi-reference electrode and shared counter electrodes was proposed. It enabled multiplexed electrochemical detection HRP and bisphenol A ^[45]. It is noteworthy that the limiting factor of inkjet printing is the lateral resolution. The lateral resolution in the range of 10−20 μm depends on the smallest drop size. It was thought that this limit generally excluded inkjet printing for the fabrication of nanoscale devices. However, inkjet printing was used to vertically stack nanoporous redoxcycling sensors. And then, printed electronics displayed the potential of promoting current redox cycling into arrays for high-throughput screening applications [39]. It is another method to miniaturize and integrate closely spaced electrode systems.

2.5.2 Digital microfluidics

Digital microfluidics (DMF) is an emerging liquid manipulation technology in whic an open array of electrodes enables control over individual picoliter- to microliter-sized droplets. can be controlled individually in time and space. Droplets serving as an isolated vessel for reaction processes, avoids cross talk between samples or reagents. At the same time, droplets on open space can be more easily controlled to merge, split and move on flexible paths than achieved with complex closed channel in microfluidic chip. Because of its unique advantages, DMF is being applied to a wide range of fields including multiplex sensoring ^[80–82].

Since the number of electrical connections limited the number and type of droplet operation in conventional EWOD device, a DMF platform with impedance sensor based on an active matrix electrowetting on dielectric (AM-EWOD) device was reported.  In this device, a thin film transistor (TFT) array replaced the patterned electrodes of a traditional EWOD device, facilitating independent control of each electrode. The fully reconfigurable arrays make multiple simultaneous operations programmed on thousands of individually addressable electrodes possible. To improve assay reliability and accuracy, this sensor has feedback, error detection and closed loop control in an assay sequence. A 64 × 64 format AM-EWOD device with impedance sensor was firstly described. After that, a colorimetric assay was implemented with this device to measure glucose in human blood serum. Comparable performance with the same assay on a microtitre plate was obtained ^[84]. A devices have 16800 electrodes was reported which could make independent multiple and simultaneous droplet operations possible. This DMF platform was used for the analysis of DNA containing the bla(CTX-M-15) gene with isothermal DNA amplification monitored in real-time using exonuclease fluorescent probes. The detection of target DNA with a detection limit of a single copy within about 15 minutes was achieved ^[83].

2.5.3 Paper-based sensors

Paper-based sensors with many unique properties including passive liquid transport and compatibility with biochemical, are a new alternative technology for fabricating simple, low-cost, portable and disposable analytical devices. Current paper-based sensors are focused on microfluidic delivery of solution to the detection site and more advanced complex 3-D geometries [85]. For multiple sensoring, a printing and modular fabrication of a paper-based digital microfluidic chip integrated with multiple electrochemical sensors (ECSs), a portable electrical control system and wireless control system was developed. The chip plate in electrodes for active electrowetting actuation of digital drops and ECSs were all fabricated by affordable printing techniques. This open–closed hybridized chip formats realized the detection of three diagnostic biological molecules (glucose, dopamine, and uric acid in human serum) ^[79].     

2.5.3 Field-effect transistors

Field effect based sensors have shown remarkable capability of small size, low cost, rapid response for real-time detection ^[86,87]. Because of the ease of collecting electrical signal transduced by electrolyte–insulator–semiconductor (EIS) with standard instrumentation and the integrated system of microfluidic chip and EIS sensor is easy to commercialize, it has received great attention recently in label-free detection of biomolecule [88]. Lin et al published a microfluidic chip incorporated with electrolyte–insulator–semiconductor sensors for measuring glucose and creatinine in blood serum. The glucose and creatinine could be detected in 60 s with a linear range from 2-8 mM and 10^-2 to 10 mM respectively which is a meaningful range in human blood ^[89]. Recently, miniaturized filed-effect transistor (FET)-based biosensors was developed by a package technology. A FET sensor array was also made by embedding eight packaged FET chips into one plastic substrate.[90].

2.5.4 Resistive pulse sensor

Resistive pulse (RP) sensing is a method for determining particles’ physical properties, including concentration, volume, charge, and shape, by analyzing the measurable change in resistance of a conducting channel as the particles pass through it. Since there is a measurable decrease in the ionic current, or a ‘pulse’ caused by each particle’s passage through the channel. RP is very powerful for analyte sensing of diverse targets, for example DNA, virus, and blood cell counting. At the same time, RP is most useful for targets below 200nm in size, in which the use of conventional optical microscopy is prohibited due to the reason of diffraction. Other advantages of RP include the ability to measure particles individually, high-throughput, low-cost setup, and low computational complexity ^[98]. An on-chip multichannel resistive pulse sensor for high throughput counting of microscale particles was demonstrated. Detection was achieved by frequency division multiplexing. Each microchannel was modulated with its own frequency. The measured signal of a pair of electrodeds was demodulated to the signal across individual channel. In addition, the ac modulation method was used in this paper to reduce the polarization effect on the microelectrodes allowing the measurement of the particle sizes with greatly reduced error ^[99].

2.7 More functionalities integrated microfluidic chip electro-sensors platform

As have been reviewed, a wealth of papers utilized microfluidics for sample transportion or reagent mixing. Most of these microfluidics simply comprised  channels with an inlet and an outlet. The emphasis on the biosensor and for the sake of reliable operation are main two reasons for a simple microfluidic structure sacrificing sophistication. Gradually there are more advanced systems where several (other) functionalities are integrated, closer to the lab-on-a-chip portable analyzer [5].

A microfluidic system capable of automatical measurement of urea and creatinine was presented. A continuous processes including sample pretreatment, regent mixing, transportation and final electrochemical sensor arrays based detection were executed on a single chip [104].  A microfluidic device capable of on-chip whole blood processing and simultaneous label-free electrical detection of multiple analytes with dual integrated complementary metal–oxide–semiconductor (CMOS) polysilicon nanowire sensors (MINS) was developed. A serpentine microchannel with dam structures was micromachined for nonlysed cells or debris trapping, uniform plasma/buffer mixing. The efficacy of on-chip whole blood processing followed by simultaneous Hb and HbA1c measurement in just 30 minutes with only 5 μL of whole blood were demonstrated [75].

Figure 8. (A) Schematic of the MINS system, consisting of (1) the PMMA microfluidic chip, (2) two piezoelectric micropumps and (3) dual CMOS polysilicon nanowire sensors. (B) The photo of the MINS platform. The overall size is 20 cm (L: length) × 15 cm (W: width) × 5 cm (H: height). (C) The photo of the PMMA microfluidic chip. The chip size is 50 mm L × 50 mm W × 4 mm H. The size of the main channel is 1 mm W × 3 mm H, and the height of the dam structure is 0.3 mm [75].

For DNA detection of pathogens, on-chip genetic amplification techniques and sample preparation via immunomagnetic separation were integrated with sequence-specific electrochemical DNA (E-DNA) sensors, which allowed the detection of influenza virus directly in throat swabs and the multiplexed detection of related bacterial strains in the blood of septic mice^[107].

Figure 9. Overview of the LAMP chip assay. (A) Unprocessed, whole blood from infected animals is introduced into the chip’s amplification chamber along with LAMP reagents and heated at 65 °C. The reaction mixture containing single-stranded amplicons is then pushed into (B) the electrochemical detection chamber. This chamber contains a duplexed electrode array that supports simultaneous, sequence-specific electrochemical detection by selectively hybridizing with amplicons from S. Typhimurium or S. Choleraesuis, (C) generating a detectable decrease in current [107].

In order to assist the spatial manipulation of particles/cells on lab-on-a-chip devices, common used microscopy negates the advantages of cost and size brought by microfluidic assays. In a microfluidic CODES, resistive pulse sensing was integrated to orthogonally detect particles in multiple channels. Three coplanar electrodes were routed to create multiple Coulter counters producing distinct orthogonal digital codes when they detected particles. As a proof of principle, this technology was used to detect human ovarian cancer cells in four different microfluidic channels fabricated through soft lithography. Together with telecomunication networks and mathematical principle to decode signals, this microfluidic CODES offers a simple, all-electronic interface that is well suited in resource-limited case ^[100,101]. Some of the most important design considerations for combining (planar) sensors with microfluidic systems are laid out and explained in a paper by Squires[109]. The interplay between channel dimensions, sensor dimensions, flow velocities, diffusional transport, advective transport and reaction rate are explored and the consequences of a bad design on the overall performance of the sensor system are also investigated by Lafleur ^[5].

Although every kind of sensor technique has its own advantages, detection based on single technology can only provide limited information. To solve the problem, several research groups have tried to integrate two or more sensors in one system. A MEMS multi-mode sensing system integrating electrochemical working electrode and solid-mounted thin-film piezoelectric resonator (SMR). This system can detect glucose concentration change with electrochemical sensor, track the concentration variation to viscosity through the impedance response of SMR^[102].

3. Microfluidic chip based Multiplex optical-sensor

As one of the largest branches of chemical and biosensor, optical biosensors is a sensing device which can analyze the chemical and biological information of analyte by absorption, reflection, fluorescence or luminescence, scattering, refraction, polarized light and other optical properties based on spectral chemistry, optical waveguide and measurement technology ^[110]. Integrating microfluidics platform with optics systems combines the advantage of lab-chip platforms with the benefits of optical technologies ^[111]. Due to the ease of interfacing microfluidic devices with the conventional optical detection instruments commonly found in laboratories (inverted fluorescence microscopes, digital CCD cameras, simple LEDs and photodiodes set-ups, and even smartphones), optical detection is ubiquitous in microfluidic applications ^[5].

3.1 On-chip fluorescence and luminescence biosensors

Fluorescence-based and luminescence-based biosensors are by far the most prevalent type of optical biosensor encountered in microfluidic applications thanks to their ease of implementation. They benefit from very low detection limits, high selectivity and the wide array of fluorescence labels available for tagging biomolecules ^[5]. Depending on whether the sensors are immobilized or not, the methods to realize fluorescence-based biosensors in microfluidic applications can be divided into heterogeneous assay and homogenous assay. Immobilized fluorescent sensors, beads based sensor array and dedicated optical setting such as lens and microspectrometer are the heterogeneous assay sensor. Different with immobilized heterogennous senors, fluorescent optical sensors floated in solution represents another homogenous biosensors. Nowadays electrochemical sensors are all carried out in heterogeneous manner. Therefore, in the mode of homogeneity is a special character owned by optical sensors in which there is no need to immobilize.

3.1.1 Heterogeneous fluorescence and luminescence biosensors

After self-assembled monolayers (SAMs) with metal ion sensing properties was immobilized on the walls of glass microchannels, five parallel combinatorial synthesis of sensing SAMs in individually addressable microchannels towards the generation of optical sensor arrays and sensing chips were then developed [112]. A supramolecular luminescent sensor platform based on self-assembled monolayers (SAMs) was implemented in a microfluidic device either. Eu(III)-EDTA complex was bound to beta-cyclodextrin monolayers via orthogonal supramolecular host-guest interactions. The self-assembly of the Eu(III)-EDTA conjugate and naphthalene beta-diketone as an antenna resulted in the formation of a highly luminescent lanthanide complex on the microchannel surface. Parallel fabrication of five sensing SAMs in a single multichannel chip was performed, as a first demonstration of phosphate and carboxylic acid screening in a multiplexed format allowing the establishment of a general detection platform for both analyte systems in a single test run with μM and nM detection sensitivity respectively ^[113]. Zhang et al also published a simple magnetic immunofluorescence assay for detection of multiple pathogens (H9N2、H1N1、H3N2) by replacing the fluorophore with the QDs. Fast analysis (a total assay time of less than 50 min) and low detection limit (H1N1 10.98 ng/mL, H3N2 13.76 ng/mL and H9N2 9.35 ng/mL) were shown. Replacing the antibodies with the capture probe, a multiple synchronous DNA hybridization analysis for detection of the nucleic acids of the three subtype AIV (H9N2、H1N1、H3N2) was structured. And this system also showed fast analysis (the whole assay time of less than 80 min) and low detection limit (H1N1 0.21 nM, H3N2 0.16 nM and H9N2 0.12 nM) ^[114]. DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. The multiple target detection of thrombin and prostate specific antigen on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor ^[115]. Qi published a novel three-dimensional (3D) origami ion imprinted polymers microfluidic paper-based chip device by grafting with CdTe QDs for specific, sensitive and multiplexed detection of Cu²⁺ and Hg²⁺ ions. The surface of the paper was activated by grafting with CdTe QDs through amino processing and formation of Cu²⁺ or Hg²⁺ IIPs and CdTe QDs complex that led to fluorescence quenching of QDs because the photo luminescent energy of QDs could be delivered to the complex. This method could realize the transfer of liquid phase of QDs@IIPs to the solid glass fiber paper and improve the portability of the device. Compared with floated QDs quenching sensor system, the immobilized QDs quenching platform allowed to simultaneous detection with good selectivity and sensitivity ^[116]. Another simple, and multi-analyte assay system was fabricated by the integration of three kinds of biochips into microfluidic device for simultaneous multi-detection of vascular endothelial growth factor, prostate-specific antigen, and PCa circulating tumor cells (CTC) in human serum for accurate diagnosis of PCa. The integrated device was able to be put in the ELISA reader for signal analysis after sample incubation, no necessary of further fluorescence staining or microscopy counting [105]

Recently, a more efficient ‘signal-on’ aptasensing strategy based on Förster resonance energy transfer (FRET) has attracted more attention. When the emission spectrum of donor fluorescent molecule and the absorption spectrum of receptor overlap, and the distance between two molecules is within 10nm, FRET happens as a phenomenon of non-radioactive energy transfer via long-range dipole-dipole interactions. This effect forms the basis of wide application of elaborate-designed FRET with pronounced selectivity and sensitivity ^[117]. A miniature multiplex chip was created for in situ detection of cancer cells by implementing a novel graphene oxide (GO)-based FRET biosensor strategy, i.e. assaying the cell-induced fluorescence recovery from the dye-labeled aptamer/graphene oxide complex. Fluorescence intensity measurement and image analyses demonstrated that this microfluidic biosensing method exhibited rapid, selective and sensitive fluorescence responses to the quantities of the target CCRF-CEM cancer cells. Seven different cancer cell samples could be measured at the same time in such a microfluidic chip by utilizing a parallel-scale homogenous detection ^[118].

3.1.2 Homogenous Fluorescence and luminescence biosensors

Compared with the heterogeneous assay sensor, homogenous optical sensors making the assays less complex, more robust, easily automated, scalable for parallel assays of patient samples, as desired in clinical and point of care applications, is also a worthy goal [119,^120]. But in the case of simultaneous multiplex sensors, floated sensors bring new challenge in discriminating signals from different sensors. If this homogenous assay is applied to simultaneously parallel multiple target sensing, multi-color probes can be used.

But the advance towards high throughput sensing is hampered by the limited number of colored probes available, not withunderstanding the difficult to readout and discriminate multi-color signal in the micro- or nanochannel without spatial separation. To address this difficulty, multichannel based multiplex sensors were then employed. For example, in order to detect two different DNA molecules simultaneously, Le published a rapid, simple and portable method for detecting the mutant types of NRAS genes codon 12 and 61 simultaneously by using bead-quantum dots based multi-channel microfluidic chip. Bead-QDs-DNA probes and dyes which can be intercalated inside the double-stranded DNA were used to detect the target DNA by the fluorescence quenching of the QDs due to FRET between QDs and intercalating dyes after DNA hybridization. It was demonstrated that this system was sensitive with low target DNA concentration of 0.1 mM and could complete detection within at less than 14 minutes ^[121]. Atalay published a novel model-based methodology based on microfluidic chips for the quantitative determination of sucrose, D-glucose and D-fructose. This chip could measure the change of concentration of the reaction product NADH by fluorescence microscope, which was stoichiometrically related to the concentration of those sugars via cascade of specific enzymatic reactions. A reduced order mathematical model was developed to help optimize the minimize sensor response time and maximize the signal output. A parallel implementation of the assays can further improve the throughput ^[122]. A simple method to fabricate a microfluidic biosensor being able to detect substrates for H2O2-generating oxidase was presented. The biosensor consists of three components (quantum dot–enzyme conjugates, hydrogel microstructures, and a set of microchannels) that are hierarchically integrated into a microfluidic device. Glucose oxidase (GOX) and alcohol oxidase (AOX) were conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning proces. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H2O2, which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50 M of glucose and 70 M of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using this microfluidic device composed of multiple microchannels ^[123].

Without the need to fabricate multichannel, another kind of separation based sensor with single label in single channel was developed by us. Two basic steps were involved in the microfluidic chip electrophoresis-based FRET multi-sensory system. The first was similar to classic FRET. FAM-labelled aptamer was assembled non-covalently onto GO. Due to the strong interaction between oligonucleotide and GO, the fluorescence of labelled FAM was quenched by FRET between GO and FAM. After the introduction of mixed analytes, the specific interaction of target protein and oligonucleotide aptamer created a relatively rigid hybrid causing the release of oligonucleotides from the vicinity of GO and restoring the fluorescence of FAM. Secondly, after the selective fluorescence recovery, chip-CE played the critical role in multiplex sensoring by separating every protein-aptamer binding pair in the mixture. This combination with chromatography based separation technique opens up a new way to high-throughput and simultaneous optical sensor. Compared with traditional GO based RET sensor, no multi-colored probes are required here to realize innovative multi-target sensing. Unlike the strategy of sensor array biochip to achieve multi-sensoring, there is no need of sensor immobilization here. With selective fluorescent recovery and fine separation, multi-sensoring can be realized without sample pre-treatment. It is extremely easy and convenient without sample pre-labelling here. There is no need to worry whether the analyte itself can be detected by UV or fluorescent detector. A high detection sensitivity with a low background noise is ensured by the super-quenching ability of GO, selective fluorescence restoring and the high separation resolution of chip-CE. At the same time, the number of targets that can be detected simultaneously is easy to be increased along with development in the separation technology of chip-CE. This methodology can be used not only in protein sensoring, drug screening, but also in multi-bio-interaction sensoring ^[124]. This novel separation and concentration based simultaneous sensor strategy was also used as a simple, rapid, and sensitive tool for simultaneous multi-drug screening ^[125].

Another micro free-flow electrophoresis separation integrated pH sensor was reported by another group. They presented a microfluidic platform that contained a micro-flow reactor for on-chip biomolecule labelling that was directly followed by a separation bed for continuous free-flow electrophoresis and had an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly is characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine ^[126,127].