1 Introduction

Biosensor development has a system used to convert the biological response to the electric signal, which is important to detect, identify and discriminate the target molecule. Sensitivity and selectivity are two main improvements with the sensor attest the high-performance, and favors the early diagnosis of diseases. There are plenty of devices have been demonstrated with improved sensing, such as surface plasmon resonance (SPR) (Gopinath 2010; Nomura et al. 2013), surface plasmon fluorescent spectroscopy (Lakshmipriya et al. 2013a), waveguide mode sensor (Fujimaki et al. 2010; Gopinath et al. 2009; Lakshmipriya et al. 2013b), Raman spectroscopy (Picciolini et al. 2014), enzyme linked immunosorbent assay (ELISA) (Lakshmipriya et al. 2016). Among these, microfluidic-assisted detection systems are more welcomed due to its fast delivery with lesser sample. Microfluidic-based devices are with the fluidic channels, predominantly made by a polydimethylsiloxane (PDMS) microfluidic layer on the glass substrate by lithography method (Luo et al. 2005). On the PDMS surface, gold or zinc oxide or other material has been coated for improved biomolecular detections (Balakrishnan et al. 2016; Fujii 2002). Fluidic channels (generally in µm scale) are narrow compatible to deliver low sample volume. Biomolecules can be immobilized on the fluidic channel and then detected by the suitable detector. Figure 1 shows the schematic representation of the general microfluidic-based device for biomolecule detection. Using microfluidic channel different diseases have been detected by the suitable probes such as DNA, RNA, antibody and aptamer (Bosco et al. 2012; Nomura et al. 2013; Gopinath et al. 2013).

Fig. 1
figure 1

a Strategy of GNP preparation on microfluidic system. b Spectrophotometric absorption of GNP. Both aggregated and dispersed GNPs are shown

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Gold nanoparticle (GNP) has widely been used and plays a vital role to improve the sensitivity in various sensors (Boca et al. 2012; Gopinath et al. 2012; Shiao et al. 2014). GNP based colorimetric assay is one of the popular assays for the detection of DNA, RNA, small molecule, and protein, aided to identify the results without involving specific equipment (Gopinath et al. 2014). The main purpose of using GNP is because of its unique characteristics such as water dispersal, biocompatibility, and easy surface modification. GNP carries surface charge and due to electrostatic attraction, GNP can capture biomolecules to be useful for controlled assembly and disassembly assays. Further, for the immobilization of biomolecules thio-linker will aid, as this linker has the ability to bond GNP. In addition, there are various shapes of GNP such as triangle, star, sphere, cube, nanorods, nanowires were shown for different applications including, biosensor, imaging, drug delivery and tumour targeting (Chen et al. 2011; Huang et al. 2009; Nehl et al. 2006; Qu et al. 2004). The combination of microfluidic and GNP is widely used in many sensors to improve the detection. In this overview, we discussed the applications of combined system with microfluidic and GNP for biosensor developments.

2 Synthesis of GNP in microfluidic system

Gold nanoparticle usually synthesized by physical and chemical methods, such as citrate method, using plasma, UV irradiation, and wet chemical method. Generally, GNP has been prepared by reducing the gold salt in the water or organic phase (Turkevich et al. 1951; Brust et al. 1994) and reduction agents can be amino acid, ascorbic acid. GNP prepared by Turkevich method is well suited for the GNP-based colorimetric and other charge-based assays due to the preparation of GNP in water phase. For these assays, GNPs are prepared by the reaction of the chloroauric acid with sodium citrate. In this method citrate used as the capping and reducing agent and carries the surface negative charges (Kimling et al. 2006). Microfluidic-based GNP synthesis was also demonstrated with the yield of large quantities in a shorter span of time and uniform distribution in sizes. Figure 1a shows the method of preparing GNP by the microfluidic delivery system (Liu et al. 2015). In this system, GNP was prepared by controlling a number of reagents, with the optimized cooling and heating in the microfluidic system. As shown in the Fig. 1a, microfluidic-based GNP synthesis includes, mixing of HAuCl4 and Na3C6H5O7 in the microfluidic channel after injection, upon passing appropriate temperatures (heating and cooling), the final product come out as GNP. In this method, 1 % trisodium citrate dehydrate was used as the reducing agent. In this easier method, it is possible to prepare the GNP in lower volume with limited manual power. The prepared GNP usually exhibits the absorption maxima at 520 nm as dispersed particles and undergo aggregation in the presence of sodium ions with the absorption maxima at ~630 nm (Fig. 1b). The size of the GNPs is completely depends on the concentration of HAuCl4 and Na3C6H5O7 and the temperature control. Hence, right choices of phases (continuous and dispersed) are needed to control the sizes of nanoparticle and their distribution. In most of the sensor applications sizes of the GNPs were in the ranges from 10 to 80 nm. For example, in the case of colorimetric assay smaller sized GNP such as 10 or 15 has been used to avoid the nonspecific binding. At the same time, in the SPR based system, 40–80 nm of GNP conjugated biomolecules has been used for the detection system. So that, the sizes of the GNP used in different sensors have with different purposes.

3 Conjugation of biomolecules with the GNP

Gold nanoparticle can be conjugated with biomolecules through electrostatic interaction, chemical functionalization, hydrophobic interaction or physical absorption. Biomolecules such as single stranded DNA, RNA, antibody and protein can easily immobilize on the surface of the GNP for various applications including imaging, biosensing and drug delivery (Nomura et al. 2013; Gopinath et al. 2012, 2014). The charged molecules can easily immobilize on the negative charged GNP. In the case of protein, amino acid composition influences the attraction to the GNP due to variation in charges. Single stranded DNA/RNA attracts GNP through electrostatic interaction, and for immobilization of biomolecules on gold surface, thiol linker has been predominantly used (Lakshmipriya et al. 2014).

4 Biosensing applications of microfluidic and GNP

The application of prepared GNP is widely applied for various fields especially in biosensing application. The above mentioned two important tools such as microfluidic and GNP are widely used in the field of the biosensor to detect various diseases causing agents using different sensors such as Raman spectroscopy, Surface plasmon resonance-based biacore system and ELISA.

4.1 Surface plasmon resonance (SPR)

Surface plasmon resonance is the very popular sensing system working according to Kretschmann configuration (Gopinath 2010), used to detect the various biomolecules from small ligand to whole cell or virus. Lei et al. (2005) have demonstrated to detect activities of living cells in the presence of trypsin by SPR sensor assisted by microfluidic platform. In this system, they used polymethyl methacrylate (PMMA) microfluidic device and reported that this device is useful to detect the cells with lower cost. Further, this microfluidic-based SPR system also helps to detect food pathogens on PDMS (poly-di-methyl-siloxane) substrate (Zordan et al. 2009). Figure 2a shows the schematic representation of microfluidic SPR system and it has two layers, the bottom layer is made up of glass, and the top layer is made by PDMS. On the PDMS surface, biomolecule was immobilized and detected by SPR. Figure 2b displays the example of surface molecular assembly on PDMS surface for the detection of biomolecules. As shown in the Fig. 2b, first GNP conjugated antigen is used to immobilize on the PDMS substrate and then detected by its partner antibody. Depends on the antigen and antibody binding the response will be changed. In the presence of GNP, the number of the capturing of antigen is higher due to the wider surface area provided by GNP. These changes are considered as the real binding for the antigen and antibody interaction. SPR combined microfluidic surface shows the higher sensitivity in SPR based biacore system is a very popular commercialized system utilizing the microfluidic delivery and helps to detect a wide range of molecules. In this system the chip used with gold coated microfluidic delivery system. Each chip consist of four fluidic channels, can possible to do the four different analyses at a time. With this system various disease biomarkers have been diagnosed. In this line, influenza virus, RNA, proteins, DNA were detected using SPR based biacore system (Gopinath et al. 2007, 2008, 2010; Yuan et al. 2011; Gopinath and Kumar 2013; Lakshmipriya et al. 2014).

Fig. 2
figure 2

a Microfluidic on PDMS substrate and b biomolecular assembly on PDMS substrate and response

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4.2 Colorimetric assay

GNP has the unique chemical and physical properties; it has the high extinction coefficient and surface plasmon adsorption. GNP is commonly used for colorimetric-based assay, which is easy and shows the visual detection of the analyte without involving special equipment. Using these GNP based colorimetric assays, we can detect assembly of RNA, DNA, protein, even the cancerous cells. In the past, a combination of GNP colorimetric and microfluidic has brought the higher sensitivity detection. In general metal ions are commonly used to detect microfluidic-based colorimetric assays. Figure 3a shows the colorimetric assay detection using metal ions such as Ca2+, Mg+, K+ and Na+. In this assay, DNA/RNA was attached on the GNP by hydrophobic/electrostatic attraction due to charged phosphate backbone of the DNA. And then when the metal ion was introduced to this complex, the color of the GNP changes to purple due to the aggregation. Figure 3b shows the diagrammatic representation of the colorimetric assay with the microfluidic system. Chen (2014) have found that mercury ions from river and pond water can be detected by GNP based microfluidic colorimetric assay with the detection limit of 50 nM, and also they performed simultaneous multiple detections. Further, Cu2+ was also detected from the tap water with the detection limit of 0.75–50 μmol/L using microfluidic-based colorimetric assay (Liu et al. 2012). Elghanian et al. (2010) detected the polynucleotides by GNP based colorimetric assay and the limit of detection was found to be 10 fM, and cysteine was also detected by the GNP conjugated DNA (Lee et al. 2008). Liu et al. (2006) detected different samples by lateral flow method assisted with GNP conjugated aptamer in colorimetric assay. They detected the small molecules adenosine, cocaine in the serum sample. Since it is important to detect the level of metal ions in the drinking water, this method is useful for quick and easier in practical application.

Fig. 3
figure 3

a Scheme for controlled assembly and disassembly of DNA on GNP. b Detection of mercury using microfluidic-GNP set-up

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4.3 Microfluidic enzyme linked immunosorbent assay (ELISA)

Enzyme linked immunosorbent assay is an important technique has been used widely to detect various kinds of diseases with suitable antibodies. Until now ELISA is one of the promising immunoassays to screen several diseases including HIV (de la Rica and Stevens 2012). Combining ELISA with microfluidic-GNP will yield more impact for the detection of biomolecules; especially it was proved with the improvement of sensitivity. Figure 4 shows the representation of microfluidic ELISA. In which, the biomolecules are injected into the microfluidic channel and detected upon binding with its antibody. In general, antigen has been immobilized on the ELISA plate surface followed by the attachment of the primary antibody. And then the suitable secondary antibody conjugated enzyme (example, horse radish peroxidase, alkaline phosphatase) was allowed to bind with the primary antibody and then finally detected by the suitable substrate for the enzyme. In this microfluidic ELISA, secondary antibody conjugated GNP was used and the signal enhancement was also shown with the silver substrate, this allows the higher sensitivity detection. Chin et al. (2011) have detected the HIV virus using the miniaturized microfluidic ELISA assisted with GNP. Apart from this, paper based ELISA system brings more advantages with the simplest form of detection. Li et al. (2010) detected the IgG using the paper-based microfluidic ELISA system with the detection limit of 3.9 fM. Microfluidic-based ELISA had also used for the imaging purposes effectively. Eteshola and Balberg (2004) used to detect the sheep IgM and found the limit of detection as 17 nM and they quantified the fluorescence from the biochemical reaction on the microfluidic ELISA by spectrofluorometer.

Fig. 4
figure 4

Microfluidic-based ELISA. Molecular assembly is shown for the detection of biomolecule

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4.4 Raman spectroscopy

Raman spectroscopy is a powerful technique in the biosensor development due to its high specific biodetection. Combination of Raman spectroscopy with the microfluidic system brings out the miniatured detection of diseases in the simplest way. Since PDMS is a high Raman active material, in general PDMS was used to prepare the microfluidic in the Raman spectroscopy. Silver or gold colloids used for the efficient detection of biomolecules by the Raman spectroscopy (Ashok and Dholakia 2012; Yang et al. 2013). Using this technique, all biomolecules (DNA, RNA, antigen and antibody), including whole cells can be detected. Pallaoro et al. (2015) have detected the cancer cells using the microfluidic-based Raman spectroscopy. They detected the prostate cancer cells with the lowest count possible (1 in the 100 cells). And also they differentiated the prostate against non-prostate cancer cells. Piorek et al. (2012) found to detect the trace amount of vapor and shown the improvement of detection by ninefold using Raman spectroscopy. Vasopressin hormone was detected by the aptamer conjugated nanopillar with the aid of microfluidic-based Raman spectroscopy. The limit of detection was found to be lower picomolar (Yang et al. 2013). Liu et al. (2014) made the paper-based microfluidic system for the detection of the crystal violet.

5 Detection of diseases by microfluidic: signal enhancement by GNP

As we already mentioned, microfluidic-based detection gives high sensitive detection with lower sample consumption in various sensors. GNP combined with microfluidic delivery system helps to detect various disease-causing agents ranged from smaller nucleic acid to the whole virus. SadAbadi et al. (2013) have detected the Bovine somatotropin from milk with the detection limit of 185 pM, as it is mandatory to detect the level of bovine somatotropin in the milk. Also, the important disease HIV was detected by this method (Chin et al. 2011), using anti-HIV antibody. Rohrman et al. (2012) have detected the HIV by the amplification of HIV-1 RNA. Influenza is also one of the life threatening seasonal diseases, need to be detected at earlier stages to prevent spreading and to treat influenza diseases. Park et al. (2009) detected the avian flu virus by microfluidic-assisted gold nanowires and nanohybrids. Prostate-specific antigen (PSA) is an important target helps to prevent the prostate cancer, and the detection system was demonstrated by Zhang et al. (2015).

6 Issues with GNP based sensors

As we already explained microfluidic-based GNP assays have been demonstrated in improving sensitivity and specificity in various sensors, however, there are some issues need to be solved to improve this system. Particularly, GNP based colorimetric assays are generally highly reliable for the detection of DNA or smaller molecules. In the case of high molecular weight proteins, the system fails to suit because of non-specific binding of the protein on the GNP surface. This nonspecific binding disturbs the result and increases the signal to noise ratio, means the protein binds on the GNP surface and prevents the aggregation of GNP even in the presence of metal ions. Figure 5a shows the evidence for the nonspecific binding of protein on the GNP surface, as proved by simple binding. In which, different proteins such as human clotting factor IX, Protein A, streptavidin, bovine serum albumin and serum were independently attached on the GNP and tested for aggregation in the presence of NaCl. It was expected that there will be an aggregation of GNP in the presence of NaCl, if there is no nonspecific binding of these proteins. On the contrary, we found a higher rate of nonspecific binding of these proteins on the GNP. These nonspecificities were different in each case and were more severe in the cases of factor IX, protein A and streptavidin. The way to solve this issue is to restrict the biofouling on the GNP using appropriate blocking materials.

Fig. 5
figure 5

a Analyses of biofouling on GNP. Different proteins (factor IX, protein A, streptavidin, bovine serum albumin and serum) are demonstrated for biofouling using different concentrations. b PEG-mediated strategy to prevent biofouling

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Generally, gold surface gives more nonspecificity with protein due to the surface electrostatic interaction between amine groups on the protein and the gold surface. It is necessary to use the right blocking agent to obtain the genuine results and to minimize the signal to noise ratio. A good blocking agent should reduce the background (signal noise ratio) at the same time it should increase the chance of the analyte binding to the ligand on the sensing surface. Previously, it was proved that Polyethylene glycol (PEG) based block-co-polymers have fulfilled these requirements (reducing non-specificity and bring out the original interaction of the analyte and the ligand). PEG is one of the inert materials used for surface modification in the biosensors. Modified PEGs such as PEG-amine, PEG–COOH are the common materials used in the sensor field to reduce the signal to noise ratio and improving sensitivity. PEG-based polymers give the proper orientation of biomolecules on the sensing surfaces, and promotes the high-performance detection (Nagasaki 2011). Lakshmipriya et al. (2013c) detected the FIX protein on amine modified SiO2 surface with the assistance of PEG-b-PAAc. They also proved that the combination of PEG-b-PAMA and N6-PEG shows the excellent detection on gold surface. It was found that PEG-b-PAMA shows the proper arrangement of thiolated DNA on gold surface and N6-PEG combined with GNP improved the sensitivity (Lakshmipriya et al. 2014). PEG-based polymer is the suitable blocking agent in the microfluidic system to bring one step ahead for the biomolecule detection.

7 Conclusions

The microfluidic device is the upcoming technology in the field of biosensor to detect the diseases with high specificity with low sample consumption. GNP is also one of the powerful particles proven in many sensing strategies to improve the sensitivity. Combination of microfluidic and GNP is become and more attractive in the biosensor field for detection the diseases with higher sensitivity. The combination of microfluidic delivery and GNP has been demonstrated with bench-top sensors to bedside analysis. Further, availability of different designs with microfluidic systems and tailored GNP sizes, still there are avenues to generate high-performance sensors. Since microfluidic system with GNP shows several applications, in near future it will demonstrate the great use in the handy application of detection systems such as glucometer and smart phone sensor. In addition, look for alternate ideal inert particle such as SiO2 is more suitable in the microfluidic system will minimize the nonspecificity. Apart from these, the limitations with microfluidic delivery system include, selection of right fluidic pattern, careful designing to avoid cross contamination and necessity of preliminary standardization especially for large scale set-up.