Kemajuan dan Tantangan Nanomaterial Fluorescent untuk Sintesis dan Aplikasi Biomedis
Abstrak
Dengan pesatnya perkembangan nanoteknologi, jenis baru bahan nano fluoresen (FNMs) telah bermunculan dalam dua dekade terakhir. Skala nanometer memberi FNM dengan sifat optik unik yang memainkan peran penting dalam aplikasinya dalam bioimaging dan deteksi yang bergantung pada fluoresensi. Namun, karena selektivitas rendah serta efisiensi fotoluminesensi rendah dari bahan nano fluoresen menghalangi aplikasi mereka dalam pencitraan dan deteksi sampai batas tertentu, para ilmuwan masih mencari sintesis FNM baru dengan sifat yang lebih baik. Dalam ulasan ini, berbagai nanopartikel fluoresen dirangkum termasuk titik kuantum semikonduktor, titik karbon, nanopartikel karbon, nanotube karbon, nanomaterial berbasis graphene, nanopartikel logam mulia, nanopartikel silika, fosfor dan kerangka organik. Kami menyoroti kemajuan terbaru dari perkembangan terbaru dalam sintesis FNM dan aplikasinya di bidang biomedis dalam beberapa tahun terakhir. Selanjutnya, teori utama, metode, dan keterbatasan sintesis dan aplikasi FNM telah ditinjau dan dibahas. Selain itu, tantangan dalam sintesis dan aplikasi biomedis juga dirangkum secara sistematis. Arah dan perspektif masa depan FNM dalam aplikasi klinis juga disajikan.
Pengantar
Pewarna organik konvensional menghadapi beberapa kesulitan dalam aplikasinya dalam biomedis karena cacat bawaannya seperti sitotoksisitas dan biokompatibilitas yang buruk [1]. Namun, munculnya nanomaterial fluoresen menunjukkan potensi besar dalam penggantian pewarna organik konvensional. Para ilmuwan telah mencurahkan banyak waktu dan upaya dalam penelitian bahan nano fluoresen, dan pencapaian yang relevan dalam sintesis dan aplikasi lebih dari sekadar menginspirasi.
Bentuk, ukuran dan struktur nanomaterial fluoresen menentukan sifat fisik dan kimianya, yang memiliki pengaruh besar pada kinerjanya. Oleh karena itu, sintesis nanomaterial fluoresen yang dapat dikontrol telah menjadi topik penelitian yang hangat. Kondisi eksperimental sintesis yang optimal berkontribusi pada ukuran, morfologi, dan stabilitas bahan nano fluoresen yang paling sesuai. Dalam beberapa tahun terakhir, banyak upaya telah dilakukan untuk meningkatkan biokompatibilitas bahan nano fluoresen dengan meningkatkan metode sintesis [2]. Ion logam biasanya didoping dengan titik karbon (CD) atau titik kuantum (QD) untuk memfungsikan permukaan bahan nano fluoresen di masa lalu. Namun, fluoresensi yang tidak efektif dan toksisitas yang mendasarinya menjadi ancaman bagi aplikasinya dalam bioimaging dan biolabeling [3]. Mempertimbangkan masalah ini, Zuo et al. melaporkan sistem pengiriman gen CD efisiensi tinggi. CD yang didoping dengan fluor disintesis dengan proses solvothermal, dan situs muatan positif untuk pengiriman gen dapat disediakan oleh polietilenimin bercabang (b-PEI) [4]. Dapat diantisipasi bahwa metode modifikasi permukaan baru akan menjadi area penelitian hotspot di masa depan.
Banyak upaya telah dilakukan untuk mengeksplorasi potensi nanomaterial fluoresen untuk aplikasi biomedis yang meliputi bioimaging, biodetection dan beberapa metode terapi, seperti yang ditunjukkan pada Gambar 1. Fluoresensi yang dapat diandalkan untuk aplikasi tergantung pada sifat fisik dan kimianya [5]. Oleh karena itu, pekerjaan penelitian untuk meningkatkan sifat-sifatnya seperti toksisitas, hidrofilisitas, dan biokompatibilitas telah menjadi bagian penting dalam mewujudkan penggunaan bahan nano fluoresen secara ekstensif di bidang biomedis. Dengan meningkatnya angka beberapa penyakit seperti kanker, ada peningkatan permintaan untuk diagnosis baru dan strategi terapi dengan akurasi dan kepatuhan yang lebih tinggi dari pasien [6]. Saat ini, doping ion logam atau non-logam dan modifikasi permukaan bahan nano fluoresen masih menjadi teknik dominan dalam meningkatkan efisiensi PL dan biokompatibilitasnya [7], dan penelitian terkait membuka visi baru aplikasi biomedis bahan nano fluoresen.
Diagram ikhtisar aplikasi biomedis dari bahan nano fluoresen
Mempertimbangkan potensi besar yang dimiliki nanomaterial fluoresen di bidang biomedis, ulasan ini menekankan pada kemajuan dan peningkatan terbaru. Para ilmuwan telah mengabdikan diri untuk fungsionalisasi permukaan nanomaterial fluoresen dan kinerjanya dalam aplikasi biomedis. Hanya dengan strategi sintesis yang dirancang dengan wajar, bahan fluoresen dapat diberkahi dengan kualitas efisiensi PL yang tinggi dan biokompatibilitas yang baik, yang penting untuk aplikasinya di bidang biomedis. Ulasan tentang sintesis dan aplikasi bahan fluoresen ini, kami harap, dapat membantu pembaca dalam memahami tren perkembangan umum bahan nano fluoresen saat ini.
Sintesis Nanomaterial Fluorescent
Titik Kuantum (Kristal Semikonduktor)
Titik-titik kuantum (QDs) adalah tempat penelitian dalam beberapa dekade terakhir karena penyerapan yang luas dan spektrum fotoluminesensi simetris, hasil kuantum yang tinggi, ketahanan yang tinggi terhadap photobleaching, koefisien kepunahan molar yang tinggi, dan pergeseran Stokes efektif yang besar [8]. Dalam hal mekanisme pembentukan QD, ketika pembawa muatan (elektron dan lubang) dibatasi oleh hambatan potensial ke daerah tertentu, semikonduktor menunjukkan efek ukuran kuantum dramatis yang mengakibatkan pergeseran spektrum penyerapan dan spektrum fluoresensi. Daerah kecil kurang dari panjang gelombang de Broglie pembawa muatan, atau setara, diameter nanocrystal kurang dari dua kali radius Bohr dari excitons dalam bahan massal [9]. Ketika pembawa muatan dibatasi oleh penghalang potensial dalam tiga dimensi spasial, QDs terbentuk, yang terutama terdiri dari atom dari kelompok II-VI (CdSe, ZnS), III-V (GaAs, InP), atau IV-VI (PbS, PbSe).
Sintesis QDs pertama kali dilaporkan pada tahun 1982 [10, 11]. Nanocrystals dan mikrokristal dari semikonduktor ditanam dalam matriks kaca. Dengan pengembangan bahan fluoresen, QD telah disiapkan dengan metode yang berbeda, seperti metode adsorpsi langsung, metode adsorpsi berbantuan linker, metode situ dan kombinasi metode preparasi sebelumnya. Kombinasi metode sebelumnya mencakup kombinasi semikonduktor yang disiapkan dengan prekursor QD dan kombinasi QD yang disiapkan sebelumnya dengan prekursor semikonduktor, di mana semikonduktor atau QD disiapkan secara terpisah [12].
Setelah serangkaian penelitian tentang sintesis QD, banyak peneliti melaporkan studi properti fluoresen QD. Bawendi dkk. mensintesis QDs dengan distribusi ukuran sempit melalui pengenalan prekursor semikonduktor seperti cadmium sulfide (CdS), cadmium selenide (CdSe), atau cadmium telluride (CdTe) untuk menyelidiki sifat optik yang bergantung pada ukuran QDs [13]. Sejak itu, CdSe menjadi komposisi kimia QD yang paling umum, dan berbagai modifikasi permukaan [14,15,16] atau cangkang anorganik pelindung [13, 17] telah digunakan untuk membuat stabilitas koloid.
Titik Karbon
Titik karbon (CD) adalah bahan nano yang muncul dalam keluarga nanokarbon dengan ukuran kurang dari 10 nm, yang pertama kali diperoleh dalam pemurnian nanotube karbon berdinding tunggal (SWCNTs) dengan elektroforesis pada tahun 2004 [18]. Perlu dicatat bahwa CD secara bertahap menggantikan titik kuantum semikonduktor dengan alasan kelarutan tinggi dalam air, sitotoksisitas rendah, fotostabilitas tinggi, emisi multiwarna yang bergantung pada eksitasi, fleksibilitas yang lebih baik dalam modifikasi permukaan, permeabilitas sel yang sangat baik, dan biokompatibilitas yang lebih baik [19, 20]. Secara umum, CD terutama terdiri dari titik-titik kuantum karbon (CQDs) dan titik-titik kuantum graphene (GQDs). Banyak metode sintetis untuk CD dengan ukuran yang dapat disetel secara luas dapat dibagi menjadi dua kelompok utama:metode kimia dan metode fisik [21].
Metode Sintetis Kimia
Metode sintetik kimia adalah yang paling umum digunakan dalam pembuatan titik karbon karena CD yang dihasilkan memiliki sifat yang sangat baik, seperti kelarutan air yang unggul, inertness kimia, toksisitas rendah, kemudahan fungsionalisasi dan ketahanan terhadap photobleaching. Secara umum, metode sintetik kimia meliputi sintesis elektrokimia [22, 23], oksidasi asam [24, 25], karbonisasi hidrotermal [26], perawatan dengan bantuan gelombang mikro/ultrasonik [27,28,29], metode kimia larutan [30], sintesis yang didukung [31], dll.
Di antara banyak metode sintetis, sintesis elektrokimia telah berulang kali dilaporkan selama beberapa dekade terakhir. Kelompok Zhao melaporkan metode baru untuk menyiapkan CD dengan sitotoksisitas rendah melalui sintesis elektrooksidasi, di mana CD dibuat melalui oksidasi elektroda kolom grafit terhadap elektroda kalomel jenuh dengan elektroda penghitung kawat Pt di NaH2 PO4 larutan air [22]. Supernatan kemudian ultrafilter melalui perangkat filter sentrifugal untuk mendapatkan CD dengan fluoresensi biru dan kuning, masing-masing. Pendekatan elektrokimia langsung lainnya baru-baru ini dilaporkan oleh Qu et al., untuk GQD dengan ukuran seragam 3-5 nm dengan oksidasi elektrokimia elektroda graphene dalam larutan buffer fosfat [23]. Warna photoluminescent (PL) dari partikel ini adalah hijau.
Mao dkk. menyelesaikan sintesis oksidasi pembakaran CD pada tahun 2007 dengan mencampur jelaga lilin dengan oksidan, diikuti dengan refluks, sentrifugasi dan dialisis untuk memurnikan CD. Spektrum fotoluminesensi CD yang disiapkan memiliki rentang warna yang luas, dengan panjang gelombang puncak emisi mulai dari 415 (ungu) hingga 615 nm (oranye-merah). Kemudian, CD yang diperoleh selanjutnya dilakukan elektroforesis gel poliakrilamida untuk memisahkan CD dengan karakterisasi optik yang berbeda. Oksidasi asam juga telah banyak digunakan untuk persiapan bahan nano yang stabil seperti titik karbon. Setelah perlakuan asam karbon nanotube/grafit dan refluks, CD yang dihasilkan dari 3-4 nm menyajikan cairan transparan cokelat yang memancarkan fluoresensi kuning cerah di bawah sinar ultraviolet dan cukup stabil dalam garam. Hal ini membuat CD dengan fluoresensi gelombang panjang (kuning/oranye/merah) memiliki penetrasi yang lebih baik. Larutan CD dapat diawetkan pada suhu kamar untuk waktu yang lama dan tidak terbentuk endapan yang menyebabkan hilangnya fluoresensi [25].
Sintesis gelombang mikro/ultrasonik secara bertahap dan terutama menjadi teknologi sintetis tambahan dalam proses sintesis [32]. CD fluoresen, berdiameter 3-5 nm, disintesis oleh kelompok Xiao melalui pendekatan bantuan gelombang mikro yang ekonomis, cepat, dan ramah lingkungan [33]. Fitur yang paling menonjol dari pendekatan satu langkah ini adalah bahwa baik pembentukan dan fungsionalisasi CD diselesaikan secara bersamaan melalui pirolisis gelombang mikro yang berasal dari cairan ionik untuk pertama kalinya [34]. Proses reaksi terjadi dalam oven microwave dengan menggunakan cairan ionik murah sebagai sumber karbon dan larutan berubah dari tidak berwarna menjadi coklat tua seiring berjalannya waktu reaksi [35]. Tang dkk. menggunakan metode ultrasonik atas dasar glukosa atau karbon aktif sebagai sumber karbon untuk mensintesis CD larut air monodispersi. Mereka memancarkan fluoresensi cerah dan berwarna-warni [28]. Senada dengan itu, Vanesa Romero dkk. memperoleh nitrogen yang sangat berfluoresensi (N) dan sulfur (S) co-doped carbon dots (CDs) setelah oksidasi fotokimia karbohidrat dalam sayuran. Co-doping N dan S meningkatkan jumlah situs aktif pada permukaan CD, sehingga meningkatkan kinerja pendarannya [36]. Titik-titik kuantum karbon yang didoping nitrogen (NCQDs), sebuah probe fluoresen, berhasil diterapkan pada penentuan doksisiklin [37]. Pathak dkk. menyiapkan titik karbon co-doped dengan nitrogen dan sulfur (NSCDs), yang disintesis dari buffer tiourea dan tris-asetat-etilendiamin dengan metode hidrotermal gelombang mikro. NSCD digunakan untuk mencitrakan berbagai bakteri patogen dan sel epitel bukal manusia karena fluorometri multiwarna [38].
Mempertimbangkan bahwa sebagian besar metode sintetik yang disebutkan di atas membutuhkan asam kuat, beberapa langkah eksperimental yang rumit, dan modifikasi lebih lanjut dengan senyawa lain untuk meningkatkan kelarutan dalam air dari CD dan meningkatkan sifat fotoluminesensinya, beberapa tim peneliti mengeksploitasi karbonisasi hidrotermal dari fotoluminesensi karbohidrat seperti kitosan, glukosa, asam sitrat, dll. untuk menghindari proses pemurnian dan fungsionalisasi yang kompleks dan memakan waktu [39]. Yang dkk. menjelaskan metode sintetik satu langkah untuk CD fluoresen dengan fungsi amino tinggi dengan hasil kuantum (QY) 7,8% dengan karbonisasi hidrotermal kitosan pada suhu ringan. Metode ini tidak membutuhkan pelarut asam kuat atau reagen pasif permukaan. Selain itu, gugus fungsi pada permukaan CD meningkatkan kelarutan dalam air dan mengurangi potensi biotoksisitasnya [26]. Titik karbon multi-doping (MCD), dengan emisi cerah dan warna-menyesuaikan, disintesis dengan metode satu pot tanpa passivasi permukaan lebih lanjut. MCD yang disintesis didoping dengan elemen biogenik yang melimpah (O, N, P) dan karenanya menampilkan emisi fluoresen yang kuat dan karakteristik yang bergantung pada panjang gelombang eksitasi, kelarutan dalam air yang baik, stabilitas optik yang tinggi, serta stabilitas ion. MCD tidak hanya dapat secara selektif dan sensitif mendeteksi Fe
3+
di bawah deteksi cahaya biru pada 15,9 nm, tetapi juga mengukur Fe intraseluler
3+
melalui pencitraan fluoresensi multi-warna [40].
Untuk metode kimia larutan, kondensasi oksidatif gugus aril telah berhasil diterapkan pada preparasi GQD selama beberapa dekade terakhir. GQD koloid stabil dengan ukuran dan struktur yang diinginkan diproduksi oleh kelompok Li dengan strategi pelarutan. Metode ini mencapai tunability ukuran dan distribusi ukuran CD yang sempit tanpa proses pemisahan ukuran yang tidak praktis [30]. Ketika datang ke prosedur sintetis yang didukung, sejumlah tim peneliti telah memanfaatkannya untuk menyelesaikan sintesis bahan nano monodispersi seperti CD berukuran nano. Kelompok Zhu mengadopsi bola silika mesopori (MS) sebagai nanoreaktor dan asam sitrat sebagai prekursor karbon dan CD hidrofilik dengan ukuran 1,5–2,5 nm disiapkan dengan metode impregnasi. CD dengan efisiensi fotoluminesensi tinggi sebesar 23% mampu memancarkan pendaran biru yang kuat dan menghadirkan sifat pendaran konversi yang sangat baik [31]. Titik karbon emisi kuning cerah (Y-CD) disiapkan oleh Yan et al. melalui metode solvotermal, menggunakan asam sitrat anhidrat sebagai sumber karbon dan 2, 3-phenazinediamine sebagai sumber nitrogen. Y-CD dengan gugus karboksil yang melimpah menunjukkan hasil kuantum fluoresensi yang terhormat (24%), pergeseran Stokes 188-nm, sensitivitas tinggi dan stabilitas yang sangat baik [41]. Metode sintetik dan properti CD disajikan pada Tabel 1.
Metode Sintetis Fisik
Secara umum, metode sintetik fisik terutama mencakup pelepasan busur, ablasi/pasifasi laser, dan perawatan plasma. Xu dan rekan kerjanya mengoksidasi jelaga pelepasan busur dengan HNO3 dan kemudian dipisahkan suspensi dengan elektroforesis gel menjadi SWCNTs. Mereka akhirnya mengisolasi pita yang bergerak cepat dari nanopartikel titik karbon berfluoresensi tinggi [18]. CD menggunakan bahan nano-karbon sebagai prekursor dan pelarut ramah lingkungan sebagai media cair disiapkan oleh Li et al. melalui pendekatan ablasi laser ringan [44]. Selain itu, Gokus dan rekan kerja mendemonstrasikan bahwa menggunakan plasma oksigen dapat menginduksi fluoresensi yang kuat menjadi graphene lapisan tunggal [45].
Nanopartikel Karbon
Nanopartikel karbon fluoresen, dengan sitotoksisitas yang berkurang, ketahanan terhadap photobleaching, dan peningkatan biokompatibilitas, semakin menarik perhatian untuk bioimaging dan aplikasi biomedis lainnya. Dibandingkan dengan ukuran titik karbon biasa dalam 1–6 nm, ukuran nanopartikel karbon lebih dari 20 nm, yang menghemat kesulitan untuk memisahkan, memurnikan, dan mengumpulkan [46]. Metode sintesis untuk nanopartikel karbon mirip dengan titik karbon, termasuk karbonisasi hidrotermal, perlakuan gelombang mikro, metode ablasi kimia, dan ablasi laser. Metode ini memiliki kelebihannya sendiri tetapi tidak dapat mengontrol ukuran nanopartikel secara efektif. Karbonisasi elektrokimia adalah metode satu langkah yang dapat mengontrol ukuran dan sifat pendaran nanopartikel karbon. Sayangnya, hanya ada sedikit substrat yang tersedia untuk metode ini. Saat ini, beberapa metode baru yang menarik telah dilaporkan seperti metode pembakaran fosfor pentoksida [47].
Dalam beberapa tahun terakhir, nanopartikel karbon yang cocok untuk aplikasi biomedis disintesis dengan metode yang dimodifikasi. Santu dkk. menyelesaikan sintesis nanopartikel karbon fluoresen merah berkualitas tinggi dengan karbonisasi terkontrol resorsinol [48]. Pendekatan ini melibatkan kopling fenol oksidatif yang terkait dengan dehidrasi untuk membentuk nanopartikel karbon fluoresen merah. Anara dkk. nanopartikel karbon fluoresen yang disintesis dengan hasil kuantum 6,08% menggunakan metode hidrotermal termodifikasi. Dibandingkan dengan metode konvensional yang memerlukan perlakuan termal lama hingga beberapa jam, metode ini mempersingkat waktu reaksi menjadi kurang dari 30 menit, mewujudkan sintesis cepat nanopartikel karbon fluoresen [46].
Tabung Nano Karbon
Tabung nano karbon satu dimensi (1D) telah menghasilkan perhatian besar di bidang biomedis berdasarkan sifat elektronik dan optiknya yang sangat baik. Karbon nanotube dapat dibagi menjadi nanotube karbon berdinding tunggal (SWCNTs) dan nanotube karbon multi-dinding (MWCNTs) sesuai dengan jumlah lapisan graphene silinder. Sementara SWCNT terdiri dari satu lapisan lembaran graphene yang digulung menjadi silinder, MWCNT terdiri dari beberapa lapisan konsentris lembaran graphene. Diameter luar nanotube karbon di bawah 100 nm, tetapi panjangnya bisa mencapai beberapa milimeter, yang mengarah ke rasio aspek yang sangat tinggi dan luas permukaan yang besar [49]. Selain itu, susunan atom karbon yang unik dalam karbon nanotube membentuk konjugasi -elektron yang kaya di luar nanotube [50]. Selain itu, nanotube karbon diberkahi dengan penyerapan yang kuat dan fluoresensi di wilayah NIR [51]. Semua karakteristik ini berkontribusi pada interaksi yang efektif dengan biomolekul, yang menjadikan karbon nanotube sebagai kandidat ideal untuk aplikasi biomedis.
Metode sintetis memiliki pengaruh besar pada diameter, panjang, struktur, kiralitas dan kualitas nanotube karbon, dan sementara itu, harus dipertimbangkan apakah metode ini dapat digunakan untuk produksi skala besar. Metode yang umum digunakan termasuk arc-discharge [52], laser ablation [53] dan deposisi uap kimia [54]. Selain itu, nanotube karbon perlu difungsikan untuk meningkatkan kelarutannya dan mencegahnya berkumpul dalam pelarut dan media biologis. Fungsionalisasi kovalen akan memperkenalkan cacat pada struktur nanotube karbon, yang menyebabkan penurunan dramatis atau bahkan hilangnya fluoresensi NIR mereka. Fungsionalisasi nonkovalen dengan molekul amfifilik seperti polimer akan mempertahankan struktur dan sifat fluoresen karbon nanotube, tetapi menurunkan QY karbon nanotube. Untuk mengatasi hambatan ini, metode baru untuk mensintesis dan memfungsikan nanotube karbon telah dilaporkan baru-baru ini. Lee dkk. melaporkan bahwa penambahan dithiothreitol, yang merupakan zat pereduksi, dapat meningkatkan QY fluoresen dari SWCNTs untuk pertama kalinya, menghasilkan fluorofor yang memiliki kecerahan yang setara dengan QDs [55]. Hou dkk. menyelidiki penambahan dithiothreitol ke SWCNT yang difungsikan dengan berbagai surfaktan. Untuk SWCNT yang dibungkus DNA dan SDS, QY fluoresennya meningkat secara signifikan, sementara pendinginan fluoresensi ke tingkat yang berbeda diamati untuk surfaktan lain [56]. Akibatnya, penambahan dithiothreitol ke DNA atau SWCNT yang dibungkus SDS adalah solusi yang layak untuk mencapai penerapan karbon nanotube dalam biomedis.
Nanomaterial Berbasis Grafena
Sebagai nanomaterial karbon dua dimensi, graphene dan turunannya telah banyak dieksplorasi untuk berbagai aplikasi biomedis seperti bioimgaing dan pengiriman obat. Nanomaterial graphene termasuk graphene nanosheet, graphene oxide (GO) dan nanosheet graphene oxide (rGO) tereduksi. Mereka memiliki luas permukaan yang tinggi dan sifat permukaan yang unik yang memungkinkan interaksi nonkovalen dengan molekul pewarna, biomolekul dan obat yang tidak larut dalam air. Banyak peneliti telah melaporkan metode preparasi graphene yang berbeda sejak disiapkan dengan sukses untuk pertama kalinya pada tahun 2004. Metode sintetik bahan nano graphene dapat diklasifikasikan menjadi dua kategori, top-down dan bottom-up.
Metode top-down melibatkan isolasi dari lapisan grafit yang ditumpuk untuk membentuk lembaran graphene, termasuk pengelupasan mekanis [57], pengelupasan berbasis pelarut [58] dan pengelupasan elektrokimia [59]. Gue dkk. secara sistematis mempelajari pengelupasan berbasis pelarut dengan bantuan ultrasound, dan menemukan bahwa gelombang ultrasonik memiliki efek pengelupasan yang baik. Mereka juga dapat mempengaruhi ukuran dan distribusi ketebalan lembaran graphene, yang memungkinkan sintesis yang dapat dikontrol. Pendekatan bottom-up melibatkan reorganisasi atom karbon menggunakan sumber karbon alternatif. Pertumbuhan epitaxial [60] dan deposisi uap kimia (CVD) [61] adalah metode sintesis bottom-up yang paling umum digunakan. Lembar GO terdiri dari banyak sp
2
domain yang diisolasi oleh gugus yang mengandung oksigen dapat disintesis menggunakan metode Hummer. Variasi ukuran sp
2
. ini domain membuat PL lembar GO berkisar antara 500 hingga 800 nm [62]. rGO diturunkan dari GO melalui reduksi kimia menggunakan zat pereduksi seperti hidrokuinon dan hidrazin. Dibandingkan dengan GO, fluoresensi rGO menunjukkan emisi pergeseran biru di wilayah UV bersama dengan pendinginan fluoresen, yang dikaitkan dengan jalur perkolasi antara kristal yang baru terbentuk sp
2
kelompok [63]. Akbari dkk. dijelaskan bahwa rasio sp
3
/sp
2
domain dalam lembar GO menentukan spektrum fluoresensinya. Oleh karena itu, GO adalah nanomaterial fluoresen yang menjanjikan pada rentang panjang gelombang yang luas di bawah derajat reduksi yang berbeda, yang dapat digunakan dalam aplikasi biomedis.
Bahan Nano Logam
Atom logam mulia menyajikan sitotoksisitas lebih sedikit dibandingkan dengan QD pada saat yang sama. Nanopartikel emas, perak dan tembaga telah mendapat perhatian yang meningkat dan diterapkan pada sejumlah besar bidang. Dalam bidang biomedis, efek mekanika kuantum dari nanopartikel emas, seperti emisi fotoluminesensi atau resonansi plasmon menjadikan nanopartikel emas (AuNPs) kandidat ideal untuk nanosensor in vivo lainnya dengan sitotoksisitas rendah [64, 65].
AuNPs telah menarik minat ilmiah yang luas berdasarkan kemudahan sintesis dan sifat uniknya, dan beragam metode sintetis telah dilaporkan. Sebagai salah satu metode yang paling penting, metode kimia umumnya dilakukan dengan memperlakukan larutan berair kloroaurat dengan zat pereduksi dengan adanya zat penstabil. Asam sitrat banyak digunakan, yang dapat bertindak sebagai penstabil dan agen pereduksi [66]. Namun, AuNP yang distabilkan dengan asam sitrat dapat mengalami akumulasi ireversibel selama pengembangan fungsionalisasi dengan ligan tiolat. Masalah ini dapat diatasi dengan membuat reaksi berlangsung dengan adanya polimer yang larut dalam air, surfaktan, atau agen capping yang membantu memberikan stabilitas yang lebih tinggi dan mencegah agregasi nanopartikel. Ukuran dan bentuk AuNPs dapat dikontrol dengan mengubah proporsi emas-sitrat, agen pengubah permukaan atau kondisi reaksi. Dengan metode emulsifikasi ultrasonik satu pot, Zhang dan rekan kerjanya memasukkan Bis(4-(N-(2-naphthyl) phenylamino) phenyl)-fumaronitrile dan AuNPs ke dalam misel untuk mendapatkan nanoprobe [67]. Yang terpenting, nanoprobe yang diperoleh, dengan potensi besar untuk diterapkan dalam pencitraan dan diagnosis bertarget tumor in vivo, memproses kapasitas pencitraan fluoresensi yang sangat baik, meskipun ada nanopartikel emas. Meskipun AuNP tidak beracun dalam kondisi eksperimental tertentu, toksisitas dan efek sampingnya perlu diperiksa secara menyeluruh [68].
Nanocluster Fluorescent Ag telah mendapat banyak perhatian karena sifat fisik dan kimianya yang unik. Proses sintesis nanocluster tersebut diklasifikasikan oleh perancah penstabil menjadi oligonukleotida DNA, peptida, protein, dendrimer dan polimer. Selain itu, literatur ekstensif telah menunjukkan beberapa sintesis hijau, seperti penerapan ekstrak batang berair D. trifoliata dan S. alba untuk mengoptimalkan kondisi persiapan [69].
Cu nanoclusters (Cu NCs) relatif banyak digunakan sebagai bahan logam mulia, tetapi sintesisnya masih langka karena kerentanannya terhadap oksidasi. Baru-baru ini, Kawasaki dkk. berhasil menyiapkan Cu NCs stabil dengan metode poliol berbantuan gelombang mikro [70]. DNA dapat digunakan sebagai cetakan untuk sintesis fluorescent Cu NCs. Mohir dkk. mengusulkan metode berdasarkan DNA untai ganda dalam larutan untuk mendapatkan Cu NCs dengan selektivitas tinggi [71]. Menggunakan sifat fluoresen Cu NCs, berhasil dimanfaatkan sebagai indikator sinyal nyala fluoresen yang efektif untuk penentuan RDX secara selektif [72].
Nanopartikel Silika
Mempertimbangkan sifat transparansi, stabilitas mekanik, ketahanan dan stabilisasi fluorofor yang tertanam, nanopartikel silika diterapkan secara luas dalam domain biologis. Misalnya, NP inti/kulit silika diterapkan untuk deteksi intraseluler Zn
2+
dan H2 PO4
−
dalam sel hidup disintesis sebagai nanosensor fluoresen “mati/hidup”. Dalam beberapa tahun terakhir, nanopartikel silika yang didoping dengan pewarna organik telah disintesis dan banyak digunakan dalam banyak aplikasi seperti biodeteksi [73]. Metode sintesis yang paling banyak digunakan untuk nanopartikel silika adalah metode Stöber dan metode mikroemulsi terbalik. Metode Stöber, pertama kali dijelaskan pada 1960-an [74], melibatkan hidrolisis alkil silikat dan kondensasi asam silikat berikutnya dalam larutan alkohol yang dikatalisis oleh penambahan amonia. Metode kedua pembentukan silika NP, metode mikroemulsi terbalik, melibatkan reaksi alkil silikat, biasanya TEOS, di dalam tetesan air dari mikroemulsi air dalam minyak [75]. Dia dkk. menyiapkan tiga jenis zat warna doping silika nanopartikel dengan metode Stöber dan metode mikroemulsi terbalik dengan menanamkan zat warna ke dalam inti partikel. Beberapa molekul secara fungsional responsif terhadap Zn
2+
diendapkan pada permukaan partikel [76]. Nanopartikel silika fluoresen digunakan untuk gambar fluoresen Zn intraseluler
2+
(H2 PO4
−
) dalam sel HeLa. Saat Zn
2+
ditambahkan ke proporsional Zn
2+
nanosensor, nanopartikel menunjukkan kemampuan untuk mendeteksi konsentrasi H2 . secara rasiometrik PO4
−
.
Secara umum, nanokomposit silika dengan monodispersitas dan biokompatibilitas yang baik dapat dengan mudah dimodifikasi lebih lanjut dengan gugus fungsi [77,78,79]. Lee dkk. nanopartikel magnetik dan pewarna fluoresen yang didoping menjadi nanopartikel silika. Nanokomposit silika ini dapat digunakan tidak hanya sebagai probe pencitraan multimodal untuk resonansi magnetik (MR) dan pencitraan fluoresensi, tetapi juga sebagai pembawa pengiriman obat antikanker [80]. Singkatnya, partikel silika bisa menjadi tempat penelitian dalam kasus seperti itu dengan penggunaan yang sangat luas.
Fosfor
Fosfor banyak digunakan dalam biomedis karena keunggulan uniknya dalam mengurangi gangguan autofluoresensi dan hamburan cahaya dari jaringan. Secara umum, fosfor terdiri dari bahan inang dan ion yang didoping [81]. Di antara bahan inang fosfor, yttrium oksida (Y2 O3 ) lebih dari menjanjikan karena fotodurabilitasnya yang rendah dan energi fononnya. Lantanida sebagian besar didoping dalam fosfor mengingat tingkat elektron dan saluran transfer energinya yang melimpah. Banyak metode telah dilaporkan untuk persiapan fosfor termasuk hidrotermal [82], pirolisis semprotan api [83], sol-gel [84], dan proses pengendapan bersama [85].
Sintesis hidrotermal muncul sebagai proses yang ideal, yang telah terbukti efisien dan ekonomis untuk mensintesis fosfor. Yu dkk. disintesis Y2 O3 :Eu
3+
fosfor dengan metode hidrotermal dengan adanya natrium sitrat [82]. Konsentrasi natrium sitrat, jumlah penambahan NaOH dan Eu dalam proses hidrotermal menentukan sifat-sifat fosfor yang diperoleh. Pirolisis semprotan api adalah metode yang menjanjikan untuk sintesis fosfor berbasis oksida yang cepat dan berurutan. Dibandingkan dengan metode konvensional, metode ini memberikan fosfor dengan kristalinitas tinggi dan distribusi dopan homogen. Khan dkk. berhasil menghasilkan Tb
3+
–doping Y2 O3 fosfor yang berdiameter sekitar 100 nm dengan distribusi ukuran yang sempit menggunakan pirolisis semprotan api [83]. Dalam metode mereka, garam alkali dicampur dengan prekursor nitrat logam lainnya, yang secara efektif mengontrol distribusi ukuran dalam kisaran yang sempit. Rute sintesis sol-gel menawarkan beberapa keuntungan, seperti homogenitas dan kemurnian yang tinggi, waktu sintesis yang berkurang, morfologi partikel yang seragam, dan distribusi ukuran partikel yang sempit [86]. Leonardo dkk. memperoleh Sm
3+
SiO yang didoping2 -Gd2 O3 fosfor dengan proses sol-gel [84]. Co-precipitation is a common and simple method for synthesizing crystalline phosphors, which ensures high homogeneity and controlled morphology characteristics. Perhaita et al. reported that the phase composition of the phosphors strongly depends on the pH during the precipitation [85].
Organic Frameworks
Covalent-organic frameworks (COFs) are new porous crystalline materials possessing outstanding stability, adsorption and low toxicity. The design of fluorescent small organic molecules with a combination of fluorescence determination methods can be used to construct more efficient nanoprobes [87]. For selective 2,4,6-trinitrophenol (TNP) determination, a novel Naphthalimide-Benzothiazole conjugate was prepared as colorimetric and fluorescent nanoprobe. The fluorescence emission peaks of receptor were selectively quenched by TNP with a limit of detection as low as 1.613 × 10
–10
M.
Metal organic frameworks (MOFs) are a kind of new generation multifunctional inorganic–organic materials with various holes and functionalized 3D crystalline structures formed by metal ions and linkers. MOFs show potential applications in separation, catalysis and other aspects due to unique attributes such as excellent chemical tenability, specific surface area and confinement of the pores. Some of the MOFs are luminescent and the quantum yield as well as light intensity will be influenced by temperature and excitation wavelength [88]. With the addition of doxycycline, Yu et al. synthesized a new functional metal–organic framework of pyromellitic acid and europium, which exhibited remarkable fluorescence enhancement at 526 nm and 617 nm. Results showed that both fluorescence intensities were positively correlated with the doxycycline concentration. The unique fluorescence response of the system could discriminate doxycycline from other tetracycline antibiotics with high selectivity.
Biomedical Applications of Fluorescent Nanomaterials
Bioimaging
Quantum Dots for Bioimaging
Fluorescent nanomaterials have been widely used in bioimaging. Compared with conventional organic fluorescent molecules, fluorescent nanomaterials are equipped with many superior properties such as high photostability, tunable emission spectra and high quantum yields [89].
As early as 1998, QDs were first successfully applied in biological imaging [90]. Since then, applications of QDs in this field have been springing up gradually. Chen’s group applied it in bioimaging and nuclear targeting with great stability and biocompatibility in living cells [91]. In spite of the extremely high sensitivity and spatial resolution of QDs, poor performances on hydrophilicity and biocompatibility hindered their applications in bioimaging in vivo. To tackle this problem, it has been found that the water-solubility of QDs can be greatly improved by attaching thiol or other hydrophilic groups to the surface of quantum dots [92]. In the same way, with the intention of improving the effectiveness and specificity of in vivo targeted imaging, targeting molecules are attached to the surface of QDs. Furthermore, the wavelength region of the emission light can be controlled by altering the size of QDs.
The combination of QDs and inorganic metal ions can optimize the application of QDs in bioimaging because the QDs’ defect-site PL peaks will be utterly removed by controlling the proportion of doped inorganic metal ions. Kuwabata, S et al. modulated the degree of Ga
3+
doping in Ag–In–Se QDs. Thus, the QDs’ defect-site PL peaks were completely removed and a sharp band-edge emission peak come into appearance [93]. They found a blue shift of the band-edge PL peak ranging from 890 to 630 nm which could be credited to the fact that the energy gap of QDs was enlarged by Ga
3+
doping. After injecting a mouse with QDs, the potential of AIGSe@GaSx core–shell QDs for bioimaging turned out satisfying. The imaging effect of this kind of QDs in mice is demonstrated in Fig. 2. However, sensing of mid-IR wavelengths is challenging due to increased dark currents and noise. HgTe QDs synthesized by colloidal method is a promising candidate for IR bioimaging by virtue of lower dark currents, higher-temperature operation, and higher detectivity [94].
Three-dimensional PL image superimposed on an X-ray CT image of the mouse subcutaneously injected with DSPC-AIGSe@GaSx liposome dispersions (each 50 mm
3
) in the back [78]
Carbon Dots for Bioimaging
The poor photostability of current fluorescent nanomaterials hinders their long-term bioimaging to a large extent. To overcome this limitation, CDs have been studied for bioimaging and some positive results have been obtained due to the great performance on PL efficiency. Enormous efforts have been put to improve their water solubility and lower their toxicity in organisms. At present, most CDs are facing a barrier in bioimaging, that is, their short-wavelength excitation disables deep penetration in tissue. Aside from this, being exposed under the short-wavelength for a long time could do irreversible damages to living cells and tissues. As shown in Fig. 3, with the purpose of overcoming this deficiency, Gao et al. designed fluorescent CDs with red emission which were successfully used for bioimaging of noble metal ions (Pt
2+
, Au
3+
, Pd
2+
) in cells and zebrafish [95]. Sun and co-workers first studied the near infrared (NIR) imaging of CDs in vivo using mice as a model. Recently, it was reported that molecules or polymers containing plentiful sulfoxide or carbonyl groups can enhance NIR fluorescence through the surface modification. As shown in Fig. 4, under NIR excitation, sulfoxide or carbonyl groups are bound to the outer layers and the edges of the CDs. Thus, electron transitions are promoted, influencing the optical bandgap [96].
A Confocal imaging of Pt
2+
in PC12 cells. (a1–e1) Bright field images. (a2–e2) Black field images of the CDs in PC12 cells with the different concentrations of Pt
2+
(0, 25, 50, 150, and 300 μM). (a3–e3) Overlay images. B Fluorescence imaging of Pt
2+
in ZF. (a1–e1) Bright field images. (a2–e2) Fluorescence images of the CDs in ZF with the various concentrations of Pt
2+
(0, 30, 60, 100, and 150 μM) [80]
Schematic of structure and energy level alignments of nontreated CDs (left column) and CDs modified with S = O/C = O‐rich molecules (right column). The red (oxygen atom) and green double‐bonded balls represent the C = O/S = O‐rich molecule [81]
Carbon Nanoparticles for Bioimaging
In the field of bioimaging, fluorescent carbon nanoparticles show unique chemical and optical properties over traditional fluorescence probes. Different size, shape and elemental composition make carbon nanoparticles with different features. The biomedical fields are always seeking the most promising fluorescent carbon nanoparticles. Gaurav et al. obtained both larger and smaller size carbon nanoparticles with laser ablation method [97]. Both green and blue fluorescence were observed in the cells incubated with the carbon nanoparticles, suggesting their different sizes. Cell viability results indicated that the prepared carbon nanoparticles were nontoxic and safe for bioimaging applications. Shazid et al. employed carbonization method to obtain fluorescenct carbon nanoparticles derived from biocompatible hyaluronic acid. Both the in vitro and in vivo bioimaging studies showed that the prepared carbon nanoparticles would be reliable and stable for opticle imaging. Moreover, based on the experimenal data, their cytotoxicity was proved to be tolerable for biomedical applications.
Carbon Nanotubes for Bioimaging
Fluorescence of carbon nanotubes in the NIR is attracting high attention for their good light penetration depth in biological tissues. However, their low quantum yield requires for considerable excitation doses, leading to a fair degree of blue-shift and failure of penetrating live tissue. Mandal et al. reported that bright and biocompatible p-nitroaryl functionalized SWCNTs, encapsulated in phospholipid-polyethylene glycol, are suitable for bioimaging applications. The prepared SWCNTs enabled high signal-to-noise ratio imaging in live brain tissues using ultra-low excitation intensities. Their 1160 nm emissions in the NIR guarantee that they will provide optimal fluorescence imaging results [98]. Ceppi et al. applied SWCNT-based fluorescence imaging to debulking surgery in an ovarian cancer mouse model. SWCNTs are coupled to an M13 bacteriophage carrying modified peptide binding to the SPARC protein, which is overexpressed in ovarian cancer, leading to real-time imaging to guide intraoperative tumor debulking. This imaging system enables detection in the NIR window with a pixel-limited resolution of 200 μm, demonstrating real potential in fluorescence imaging guided surguries for patients [99].
Furthermore, fluorescent moieties can be conjugated by a carbon nanotube backbone, which integrate strong fluorescent ability with robust mechanism strength, exhibiting ideal bioimgaing results. Katharina et al. functionalized SWCNTs with an amphiphilic C18 -alkylated polymer conjugated with bright perylene bisimide fluorophores. The polymers wrapping around the SWCNT backbones not only increase their water dispersibility but also promote their biocompatibility by providing a shield. In vitro studies on HeLa cells demonstrated that the biocompatibility of SWCNTs is dramatically improved. In microscopy studies, direct imaging of the SWCNTs' cellular uptake via perylene bisimide and SWCNT emission proved their potential for bioimging [100]. Park et al. combined carbon nanotubes with mussel adhesive proteins which can be specifically targeted at tumors in tissue. They then made carbon nanotubes conjugated with a ZW800 NIR fluorophore to obtain NIR fluorescence imaging [101]. The prepared carbon nanotube probes react with a specific tumor in one hour and can be easily eliminated via urine, demonstrating great value as tumor imaging and detecting agent.
Graphene-Based Nanomaterials for Bioimaging
Large surface area and feasible further functionalization make graphene-based nanomaterials a promising candidate for biomedical applications. However, as a result of their chemical sturctures, graphene nanosheets lack photoluminescence and rGO only display weak fluorescence, which makes it difficult to be utilized in bioimgaing applications. Many researchers attempted to resolve this problem by conjugation of fluorescent dyes and probes onto the large surface of graphene and its derivatives. Sun et al. reported an assembly strategy to prepare fluorescence probe RACD functionalized a single layer GO via π-π interaction and hydrogen bonding. The resluting nanomaterials exhibited that the fluorescent probes reduce the aggregation degree and acquire very well monodispersion, hydrophilicity and photostability, which is attributed to the strong synergy between RACD and GO [102]. Even so, fluorescence quenching remains a critical issue for these materials. In addition, the biocompatibility and toxicity of polymers applied to connect graphene-based materials and fluorescent moieties have not been adequately investigated. These facts appeal for alternative solutions to utilize graphene and its derivatives in bioimaging applications. Georgia et al. developed intrinsically photoluminescent graphene derivatives that show desirable biocompatibility and tunable fluorescence properties [103]. They can be organophilic or hydrophilic with different amine functionalization dodecylamine and hexamethylenediamine, respectively. The intrinsic fluorescent graphene-based nanomaterials possess great potential in a variety fileds of bioimgaing.
Metal Nanomaterials for Bioimaging
In recent years, fluorescent metal nanoparticles have shown great potential in bioimaging for improved disease diagnosis and treatment [104]. Gold is the most commonly used metal for bioimaging. The surface of AuNPs can be easily modified with various biomolecules such as peptides, proteins, antibodies, enzymes, and nucleic acids. These biomolecules can interact with specific cells or organelles in vivo, which makes it possible for AuNPs to be used for targeted optical imaging. Gao et al. reported a real-time in situ imaging of nucleus by AuNPs fabricated with bifunctional peptides constructed with both Au-binding affinity and nucleus-targeting ability. The bifunctional peptides showed strong binding affinity toward AuNPs and ensured good surface coverage of the nanoparticles, which made it stable and efficient for precise bioimaging of the nucleus in cells [105]. The Au-Se bond is considered as a better candidate than the Au–S bond to link the peptides and AuNPs due to the stronger ability against interference of intracellular thiol. Pan et al. prepared the Au-Se-peptide nanoprobes through a direct freezing process. The obtained nanoprobe was successfully applied to identify autophagy and apoptosis in chemotherapeutic drug treated cancer cells [106].
As a novel fluorescent imaging technology, DNA-templated silver nanoclusters (DNA-Ag NCs) have aroused the attention of many scientists due to their unique properties, especially the tunable fluorescence emission range relying on DNA sequences. However, the highly negatively charged DNA backbones have always been a great obstacle for the expansive applications in bioimaging because of poor stability as well as poor cell permeability in physiological environment. It is also noteworthy that the PL property and fluorescent efficiency of DNA-Ag NCs are far from satisfying. As a result, figuring out how to neutralize the negative charge on the surface of DNA strands is of great urgency for researchers. Recently, Lyu and co-workers successfully modified fluorescent DNA-Ag NCs with cationic polyelectrolytes via electrostatic force between the positively charged polyelectrolytes and the negatively charged phosphate groups of the DNA strands, leading to a threefold fluorescence intensity enhancement [7] (Fig. 5). Li et al. reported a facile strategy to make gold nanoclusters with positive charge and silver nanoclusters with negative charge form aggregates by electrostatic interactions. An incredible 40-fold fluorescence intensity enhancement was obtained. Results demonstrated that the physiological stability improved a lot and the cell permeability was also enhanced, which promises its practical applications in the future.
Formation of FL DNA–Ag NC–Cationic Polyelectrolyte Complexes for Cell Imaging [7]
Silica Nanoparticles for Bioimaging
Dye-doped fluorescent silica nanoparticles emerge with great potential for bioimaging as a novel and ideal platform for the monitoring of living cells and the whole body. The outer silica shell matrix protects fluorophores from outside chemical reaction factors as well as provides a hydrophilic shell for the inside insoluble nanoparticles, which renders the enhanced photo-stability and biocompatibility to the organic fluorescent dyes. Benefiting from the robust structure of silica matrices, dye-doped fluorescent silica nanoparticles have been presented with several superior properties including good biocompatibility, hydrophilic features, and high fluorescence intensity [107].
Jiao et al. also constructed a local hydrophobic cage in dye-doped fluorescent silica nanoparticles to improve their optical properties, which solves the problems of aggregation-caused quenching (ACQ) and poor photostability in aqueous media by organic fluorescence dyes benefiting from the robust structure of silica nanoparticles [108]. In addition, compared with free dyes, the fluorescent intensity both in water solution and living cells demonstrated a 12.3-fold enhancement due to the limitation of molecular motion, indicating a significant development for silica nanoparticles in biomedical applications. QDs have been developed for bioimaging both in vivo and vitro owing to their excellent optical qualities. However, a critical obstacle faced in QDs’ application in vivo is their poor biocompatibility. Inspired by the organic dye-conjugated silica-NPs, QDs-embedded silica-NPs have also been invented with the advantage that the excellent optical qualities of QDs can be retained, while the silica-NPs coat improves their biocompatibility to a large extent simultaneously. Darwish et al. reported that many QDs could be assembled around a central silica nanoparticle to form supra-NP assemblies. It was expected to be used for enhanced bioimaging because of their higher sensitivity and superior signal-to-background ratios [109]. There is reason to believe that silica-NPs conjugated with fluorescent nanomaterials with ideal optical properties will still be the dominant research interest in the future.
Phosphors for Bioimaging
For bioimaging, the sizes of phosphors need to be controlled so that they are small enough to be integrated with living cells. Furthermore, the aggregation of particles should be avoided for biocompatibility. Hence, the control of both particle sizes and dispersity in an aqueous solution is essential for the bioimaging application of the phosphors. Atabaev et al. prepared Eu, Gd-codoped Y2 O3 phosphors which had a spherical morphology within the range 61–69 nm. Enhanced PL emission and low toxicity made these phosphors suitable for bioimgaing applications [110].
Upconversion nanomaterials are able to convert lower-energy near-infrared photons to higher-energy ones as emission. This anti-Stokes photoluminescence process will lead to low background noise, large tissue penetration depth, and low photo-damage in bioimaging applications [111]. Lanthanide-based phosphors are able to show upconversion emission owing to their photodurability and low phonon energy. Nallusamy et al. reported a NIR–NIR bioimaging system based on Er
3+
:Y2 O3 phosphors by using NIR emission at 1550 nm under 980 nm excitation, which can allow a deeper penetration depth into biological tissues than ultraviolet or visible light excitation [112]. In addition, the surface of Er
3+
:Y2 O3 was electrostatically PEGylated to improve the chemical durability and dispersion stability under physiological conditions. Thakur et al. synthesized Ho
3+
/Yb
3+
co-doped GdVO4 phosphors via a modified sol–gel method. The prepared phosphors showed brilliant red upconversion emission under NIR excitation, which may be useful in bioimaging of the biomolecules [113].
Organic Frameworks for Bioimaging
Careful selection of MOF constituents can yield crystals of ultrahigh porosity and high thermal and chemical stability, with some of them being luminescent [114]. Recently, Sava Gallis’s group described a novel multifunctional MOF material platform which showed a wide spectral region from 614 to 1350 nm covering the deep red to NIR region. Both porosity and tunable emission properties made them highly suitable for in vivo bioimaging [115]. What’s more, to overcome the obstacle of MOF’s low selectivity towards malignant tissues, Liu et al. developed a target-induced bioimaging by conjugating DNA aptamers using ZrMOF nanoparticles as quenchers [116]. Based on the quenching of ZrMOF nanoparticles, target-induced bioimaging is achieved upon binding with the target.
Biodetection
Since fluorescent nanomaterials can amplify the fluorescent signals significantly and be compatible with organisms, there is increasingly more research on their application in the rapid detection of biomolecules [117]. It will shorten the analysis time to a large extent if we are able to establish a real-time detection system by fluorescent nanomaterials. It has been discovered that multiple detection can be achieved by using QDs probes simultaneously [118, 119].
Pathogen Detection
Pathogens have been an unignorable threat to human health for centuries and these include many types of microorganisms ranging from bacteria (pathogenic Escherichia coli, Salmonella, and Streptococcus pneumoniae ) and viruses (Coronavirus, Influenza virus and hepatitis virus ). However, conventional methods for pathogen detection still need improvement of detection limits and detection speeds. For their applications in detecting pathogens, Tan and co-workers reported a bioconjugated nanoparticle-based biodetection for in situ pathogen quantification, which cost less than 20 min [120]. Tan’s success promises that quick and convenient pathogen detection is possible and can be achieved with these ingenious nanomaterials in the future. Here, we list diverse pathogens detected by fluorescent nanomaterials as shown in Table 2 [119, 121,122,123,124,125,126,127,128,129].
Nucleic Acid Detection
Apart from pathogen detection, fluorescent nanomaterials have also aroused more and more interest of scientists in the detection of DNA. Owing to the merits that a number of biomolecules can be attached to the surface of fluorescent nanomaterials, the signal intensity of fluorescent nanomaterials in DNA detection can be enhanced significantly. Tan and co-workers developed an DNA detection method to detect gene products using bioconjugated dye-doped fluorescent silica nanoparticle with high sensitivity and photostability [130]. Although the analysis of nucleic acid has been successfully achieved by real-time nanomaterial fluorescence systems, there are still many shortcomings such as complex procedures or expensive instruments. To address these disadvantages, Wang’s group introduced a highly sensitive and visualized detection of nucleic acid by the combination of strand exchange amplification (SEA) and lateral flow assay strip (LFA) [131]. The system, which is possible to be widely used in areas requiring limited resource, is mainly characterized by integrating SEA with LFA (Fig. 6). There is no denying that the extremely high fluorescent signal for bioanalysis plays an irreplaceable role in these applications.
Schematic illustration of the SEA-LFA strip for the detection of nucleic acids [110]
Drug Detection
In the field of drug analysis, a facile and low-cost analytical method is always in demand for high-speed detection of specific pharmaceutical compounds. Real-time detection of drugs can be achieved with selective and sensitive fluorescent nanomaterials owing to their outstanding optical properties. In the past decade, the modification of nanomaterials has lowered their detection limit and improved their detection accuracy significantly. Recently, it is reported that ampicillin can be detected in serum sample based on aptamer, its complementary strand (CS) and gold nanoparticles (AuNPs) [132]. The limit of detection (LOD) of this method can be as low as 29.2 pM. However, there are still many limitations in the detection of drugs or target molecules in vivo. Due to low selectivity, conventional fluorescent nanomaterials inevitably generate false positive results and adverse effects in vivo. In addition, current tracking systems can hardly realize real-time tracking because of insufficient labels and excitation sources. Considering the above limitations, a new method using up/down conversion (UC/DC) PL nanomaterials has attracted increasing attention. Seo et al. reported a single-photon-driven UC/DC system which demonstrated outstanding performance in the detection of heavy metal ion (i.e. Hg
2+
) in mussels [133]. LOD of the nanohybrids was ca. 1 nM. This system is appealing to researchers in the field of fluorescent nanomaterials for biomedical applications.
Drug Delivery
Until now, the technology of treating cancers with high efficiency and targeting function is not perfect enough. Under most circumstances, the anticancer drugs are distributed and released extensively in the body, which endangers the healthy cells and tissues irreversibly. Currently, a large variety of carriers for drug delivery have been designed. However, we can hardly supervise the distribution and result of the whole delivery process. Benefiting from the recent development of the surface modification technique, fluorescent materials capped with polymers like polyethylene glycol (PEG) can bond with drugs strongly and firmly. Then, the loaded drugs will be released in response to certain conditions such as pH, osmotic gradient and the surrounding environment. However, it should be confirmed whether the drugs are transported to the specific site or not. It’s also necessary for us to consider more details such as how much of the drugs is released in different positions. Aside from being drug carriers, fluorescent nanomaterials can also demonstrate the consequences of intracellular uptake due to their fluorescence property. QDs have been applied to monitor some important properties, such as delivery efficiency, release rate and distribution of drug molecules in vivo, which are beneficial for scientists in order to understand the specific targeting pathways of drug delivery within living cells. Duan and co-workers reported a facile pH-responsive fluorescent CDs drug delivery system [134]. Loaded with dox which is effective for gastric cancer, intracellular drug delivery and tracking could be simultaneously realized in patients (Fig. 7). The report highlighted the ability of fluorescent CDs to label and track the drug delivery process for at least 48 h, which showed a great potential in bioimaging, biolabeling and traceable drug delivery. Duan et al. designed a pH and receptor dual-responsive drug delivery system [135]. Hyaluronic acid was covalently attached to the surface of CDs, and doxorubicin was loaded by electrostatic self-assembly. In the tumor microenvironment (pH 5.6), the drug is released rapidly from the drug delivery system, while in the normal physiological environment (pH 7.4), the drug is hardly released. Endocytosis occurs when the drug delivery system reaches CD44 which is a receptor rich in tumor cells and can bind specifically to the hyaluronic acid. In addition, carbon nanotubes can be used for drug delivery by virtue of their high loading efficiency. Strong π-π interactions play a critical role in binding therapeutic agents with carbon nanotubes, which can be broke through changing external conditions, resulting in the release of drugs in specific position. Pennetta et al. functionalized single and multi-walled carbon nanotubes with a pyrrole derived compound to form a doxorubicin stacked drug delivery system. Biological studies showed that the synthesized nano-conveyors can effectively deliver the drug into cell lines and improve the therapeutic effects of doxorubicin [136].
Schematic illustration of the preparation (a ) and cellular uptake (b ) of the CDs-DOX drug delivery system [113]
Photodynamic Therapy
Photodynamic therapy (PDT) is a novel therapy method for tumors which utilizes the interaction between light and photosensitizer. In PDT, reactive oxygen species (ROS) is produced from oxygen by photosensitizers in the condition of specific wavelengths of light (mostly in the area of near infrared light). The specific mechanism is presented in Fig. 8. ROS includes singlet oxygen, superoxide radicals, hydrogen peroxide, and hydroxyl radicals that possess strong cytotoxicity which cause significant destruction of tumor cells. However, there exist many defects such as limited penetration depth [137], hydrophobic properties [138], photobleaching [139], complicated procedure [140], and tumor hypoxia. PDT agents can hardly be dissolved and they will disperse extensively in vivo once they are taken, making it impossible to be targeted and selected. Fluorescent nanomaterial based photodynamic therapy developed fast in recent years [141]. Combined with the unique properties that QDs possess, such as high fluorescent efficiency and great spectral resolution, the effect of PDT can be enhanced. Barberi-Heyob, M and coworkers significantly enhanced the photodynamic efficiency with a concentration of 8 nM because of the light dose-dependent response [142]. In addition, photodynamic therapy can sometimes do harm to the skin and eyes of patients due to its photosensitive side-effect. To alleviate these adverse effects, a novel nanoparticle-based drug carrier for photodynamic therapy is reported which can provide stable aqueous dispersion of hydrophobic photosensitizers. Meanwhile, the key step of photogeneration of singlet oxygen was preserved, which is necessary for photodynamic action [143]. It is obvious that QDs combined photodynamic therapy will replace the conventional PDT someday.
Schematic illustration of producing reactive oxygen species (ROS) for the photodynamic therapy (PDT) [119]
Challenges
Synthesis Challenges
Achieving Uniform Distribution
In the synthesis process, the diameter and size distributions of FNMs can be hardly distributed uniformly due to the agglomeration of small particles. This could be fatal to the optical properties of FNMs in biomedical application. For this reason, the applications of FNMs are still at the laboratory scale. It has been confirmed that the surface properties primarily determine the agglomeration state of the nanoparticles and their size. Therefore, surface modification is promising to achieve uniform distribution of FNMs by altering their surface properties [144]. To date, silanized QDs have been widely used because the polymerized silica coating increases the stability in buffers under physiological conditions [145]. Carbon dots synthesized by hydrothermal reaction using water-soluble base were reported to be difficult to control the size and distribution of grain boundary [146]. Khanam et al. reported a facile and novel synthetic method for the preparation of hydroxyl capped CDs using an organic base and a surfactant (Triton X-100) to modify the surface. A narrow particle size distribution at 7.2 nm was found in Raman and DLS studies, which is smaller than the majority of the particles falling within the range of below 10 nm in diameter [147].
Fluorescence Quantum Yield
Fluorescence quantum yield plays a crucial role for FNMs in their efficiency for on-demand light emission. Tunable and highly fluorescent CDs can be prepared with the surface functionalization approach. Nitrogen-doped FNMs are reported to have improved fluorescence quantum yield. With increasing nitrogen content, fluorescence quantum yield can be increased to as high as 56% at high synthesis temperature [148]. A facile strategy was also developed to tune the photoluminescent properties of CDs using a microwave irradiation, with citric acid and nitrogen-containing branched polyethyleneimine (b-PEI) as precursors. At intermediate levels of b-PEI, the CDs produced a high photoluminescence yield [149]. Lin et al. explored carbon dots with a high-fluorescence quantum yield rate synthesized from L-cysteine and citric acid by the microwave-assisted method. The obtained carbon dots exhibited a high-fluorescence quantum yield (up to 85%), which is due to the combination of amidogens and sulfydryl with carbon dots, and henceforth bringing the improved fluorescence property [150]. The above examples demonstrate that nitrogen or other electron-rich atoms like sulphur can obtain satisfying fluorescence quantum yield.
Aggregation-Caused Quenching
Fluorescent molecules can emit light with high efficiency in dilute solution. However, in concentrated solution or solid state, their fluorescence will be weakened or even disappear. This phenomenon is called Aggregation-Caused Quenching (ACQ) [151]. This problem has been puzzling scientists for almost 150 years, thus hindered the extensive application of fluorescent dyes.
In order to make effective use of fluorescent dyes, scientists have attempted many methods. Most of them focused on reducing the concentration of fluorescent dyes to prevent ACQ effect. Tang et al. discovered the phenomenon of Aggregation-Induced Emission (AIE) [152]. Based on rationally designed molecules, the fluorescence of organic molecules in solid state can be attained. Still, for more than one hundred thousand different fluorescent dyes in the world, the problem of ACQ has not been completely resolved. As long as they aggregate together, ACQ will make them lose their fluorescent properties.
It is almost impossible for high concentration or solid state FNMs to show reliable fluorescence due to fluorescence quenching. Although fluorescence quenching can be used as a sensitive signal to indicate substrate concentration in analytical chemistry, [153] in the most circumstances, however, fluorescence quenching is undesirable for FNMs because it always has considerable influence on bioimaging and biodetection. To overcome this long-standing problem, Benson et al. reported a universal solution with the discovery of a class of materials called small-molecule ionic isolation lattices (SMILES) [154]. SMILES are simple to make by mixing cationic dyes with anion-binding cyanostar macrocycles. We draw inspiration from their findings and believe that similar results can be obtained if we replace cationic dyes with cationic modified FNMs.
Application Challenges
Drawbacks of UV Light FNMs
Although FNMs realized the great-leap-forward from in vitro imaging to in vivo imaging, the emission fluorescence of most of FNMs is distributed in ultraviolet region or short wavelength visible region, which limits the optical imaging in living organisms. Moreover, use of UV light for monitoring living processes in cells and tissues has some potential drawbacks as long‐term irradiation of living cells may cause DNA damage and cell death. Therefore, the development of FNMs in near-infrared region is urgently needed in the future. Although NIR FNMs have deep tissue penetration, NIR detectors and filters are needed as the excitation and emission wavelengths are too close to each other, which restricts their range of application.
Interference in Biological Environment
Almost all biological tissues will produce significant autofluorescence under short wavelength, UV and visible light radiation [155]. Autofluorescence reduces the signal‐to‐background ratio and often interferes seriously with the visual effects. Some substances in the substrate of biological tissues also have great influence on the fluorescence, which reduces the selectivity of FNMs significantly. Until now, although the application of FNMs in mice showed acceptable outcomes, it is still difficult to achieve similar results in larger mammals. Much higher luminous efficiency under low power density excitation is required to avoid the background signal interference. Furthermore, temperature and pH conditions of the biological environments strongly affect the fluorescence of some substances as well. Therefore, satisfying fluorescence of FNMs at 37 °C and physiological pH should be guaranteed. It's worth noting that the pH in tumor is lower than normal tissues. Hence, fluorescence with high selectivity in acid environment will improve the efficiency of FNMs.
Biocompatibility
Biocompatibility refers to materials or systems that are nontoxic, safe and not causing physiological or immunological reactions. QDs with unique quantum confinement effect and electro-optical properties are attractive for biomedical applications. However, toxic effects of traditional semiconductor QDs made of heavy metal ions have serious safety concerns for their undesired environmental or health effects. In the purpose of circumventing this problem, core–shell structure modification of QDs by using biocompatible ligands or polymers is one way to effectively minimize toxic effects of traditional QDs. Furthermore, scientists are searching for heavy metal-free QDs formulations. Non-toxic or less toxic carbon dots and silica nanoparticles have shown their potential as the ideal FNMs for biomedical applications. Impurities brought in the process of syntheses may influence the biocompatibility of fluorescent nanomaterials. In order to reduce the influence of toxic impurities, green synthesis methods have been arousing the interest in biomedical fields. Chowdhury et al. utilized cacao extract which is a natural product as a reducing and stabilizing agent in the synthesis of gold nanoparticles [156]. Oxalic acid, as a constituent of cacao, can reduce Au
3+
in HAuCl4 to metallic gold and stabilize the resultant nanoparticle colloidal solution. In vitro studies suggested that the cacao derived gold nanoparticles are biocompatible and suitable for biomedical applications. For MOFs, appropriate metal ions and ligands must be selected to lower the toxicity. Wang et al. employed Fe, Ti and Zr as constituents of MOFs, which are harmless and even beneficial elements to the body [157]. In vitro studies indicate that the proposed material has good biocompatibility and safety in biomedical application. What’s more, it is necessary to consider whether the difference in composition, surface charge, or modified group will have different biological effects. Taking these factors into account, we can improve the biocompatibility of FNMs with rational design.
Conclusions
Benefiting from the unique properties of fluorescent nanomaterials, some limitations and barriers of conventional materials and methods in biomedical applications can be broken through. In this review, we comprehensively present the synthesis methods and applications of fluorescent nanomaterials. The advanced synthesis methods can offer us the fluorescent nanomaterials with ideal morphology, size ranges and structures. Meanwhile, the more convenient syntheses can lower the manufacturing cost of fluorescent nanomaterials, which is critical to their widespread applications in biomedical fields. Based on the improved synthesis techniques, the performance of fluorescent nanomaterials is bound to leap in their applications. With the development of fluorescent nanomaterials, bioimaging, biodection, drug delivery and photodynamic therapy will be more widely applied in the diagnosis and treatment of diseases. Finally, challenges in synthesis and biomedical applications point out exiting questions and developing direction. We hope that this review can bring some new insights to the development of fluorescent nanomaterials.
Availability of data and materials
All data and materials are available without restrictions.
